Terapia fotodinâmica associada a quercetina microemulsionada como alternativa para o controle de infecções causadas por Staphylococcus aureus resistentes a meticilina

Abstract

Objectives: The study aimed to evaluate the antimicrobial effect of quercetin combined with antimicrobial photodynamic therapy (aPDT) and its impact on mice intradermally infected with methicillin-resistant Staphylococcus aureus (MRSA). Additionally, the study involved the standardization of a microemulsion containing quercetin and the assessment of its antimicrobial efficacy after TFA application for MRSA inactivation. Materials and Methods: Initially, in vitro studies were conducted to evaluate the antimicrobial photodynamic potential of quercetin. For this purpose, scanning spectrometry was performed to determine the wavelengths capable of activating quercetin. Subsequently, reference strains of Methicillin-Resistant Staphylococcus aureus (MRSA ATCC 43300) were exposed to various concentrations of quercetin: 100 µg/mL, 300 µg/mL, 400 µg/mL, and 500 µg/mL, and stimulated by blue LED light. The production of singlet oxygen and the zeta potential of bacteria exposed to quercetin were then evaluated to characterize a potential site of action of quercetin on the bacteria. Finally, cytotoxicity analyses were performed on nucleated HUVEC cells. Following these steps, the antimicrobial effects of quercetin as a photosensitizer against S. aureus were evaluated in vivo using a murine model of intradermal infection over 72 hours and 17 days. At this stage, 48 Balb/c mice were intradermally infected in their ears with 1.5x108 colony-forming units of MRSA 43300. After infection, they were divided into four groups (12 animals per group) (1) treated with the vehicle, (2) quercetin alone, (3) blue LED light alone, or (3) with the aPDT protocol (quercetin + blue LED light). Lastly, a microemulsion was developed as a transdermal delivery system for quercetin, with the characterization of the microemulsion system, accelerated stability testing, and antimicrobial evaluation against the MRSA 43300 strain. Results: The scanning spectrophotometry analysis showed a pronounced absorption peak when exposed to blue light in the 400 nm to 450 nm range. This characteristic enabled the conduction of in vitro tests, which revealed significant photodynamic activity against MRSA at concentrations starting from 100 µg/mL. The compound showed an indication of singlet oxygen production by the decrease in uric acid absorption and an indication of action on the bacterial cell wall through the zeta potential test. Furthermore, quercetin did not exhibit toxicity in HUVEC cells before or after photoactivation. In addition, in vivo observations indicated that aPDT with quercetin reduced the bacterial load in the draining lymph node after 72 hours. Despite its efficacy in reducing the bacterial load in the lymph node, the lesion area was more extensive in the groups treated with quercetin, with increased recruitment of polymorphonuclear cells 17 days post-infection. The animals that received treatment with quercetin exhibited immunomodulation with a strong interaction between TNF-α and IL-12p70. Finally, the microemulsion containing quercetin presented a translucent liquid appearance and characteristic particle size, polydispersity index, and zeta potential values of a microemulsion, as well as good stability after 90 days and inhibitory activity against bacterial growth at concentrations of 0.312 mg/mL when photoactivated by blue LED light. Conclusions: Quercetin appears to be a promising photosensitizer in Antimicrobial Photodynamic Therapy (TFA) demonstrating effective photodynamic activity when activated by blue LED light and inducing the death of Staphylococcus aureus both in vitro and in vivo in a murine model of intradermal infection. Furthermore, developing a microemulsion system containing quercetin also stands out due to its favorable physicochemical characteristics, long-term stability, and effective antimicrobial activity against MRSA. This system can develop therapeutic protocols without needing a local photosensitizer injection, reducing associated pain and discomfort and thus potentially decreasing treatment abandonment.