DNA-aptamer-nanographene oxide as a targeted bio-theragnostic system in antimicrobial photodynamic therapy against Porphyromonas gingivalis.

The aim of this study was to design and evaluate the specificity of a targeted bio-theragnostic system based on DNA-aptamer-nanographene oxide (NGO) against Porphyromonas gingivalis during antimicrobial photodynamic therapy (aPDT). Following synthesis and confirmation of NGO, the binding of selected labeled DNA-aptamer to NGO was performed and its hemolytic activity, cytotoxic effect, and release times were evaluated. The specificity of DNA-aptamer-NGO to P. gingivalis was determined. The antimicrobial effect, anti-biofilm potency, and anti-metabolic activity of aPDT were then assessed after the determination of the bacteriostatic and bactericidal concentrations of DNA-aptamer-NGO against P. gingivalis. Eventually, the apoptotic effect and anti-virulence capacity of aPDT based on DNA-aptamer-NGO were investigated. The results showed that NGO with a flaky, scale-like, and layered structure in non-cytotoxic DNA-aptamer-NGO has a continuous release in the weak-acid environment within a period of 240 h. The binding specificity of DNA-aptamer-NGO to P. gingivalis was confirmed by flow cytometry. When irradiated, non-hemolytic DNA-aptamer-NGO were photoactivated, generated ROS, and led to a significant decrease in the cell viability of P. gingivalis (P < 0.05). Also, the data indicated that DNA-aptamer-NGO-mediated aPDT led to a remarkable reduction of biofilms and metabolic activity of P. gingivalis compared to the control group (P < 0.05). In addition, the number of apoptotic cells increased slightly (P > 0.05) and the expression level of genes involved in bacterial biofilm formation and response to oxidative stress changed significantly after exposure to aPDT. It is concluded that aPDT using DNA-aptamer-NGO as a targeted bio-theragnostic system is a promising approach to detect and eliminate P. gingivalis as one of the main bacteria involved in periodontitis in periopathogenic complex in real-time and in situ.

Quorum quenching of Streptococcus mutans via the nano-quercetin-based antimicrobial photodynamic therapy as a potential target for cariogenic biofilm.

BACKGROUND: Quorum sensing (QS) system can regulate the expression of virulence factors and biofilm formation in Streptococcus mutans. Antimicrobial photodynamic therapy (aPDT) inhibits quorum quenching (QQ), and can be used to prevent microbial biofilm. We thereby aimed to evaluate the anti-biofilm potency and anti-metabolic activity of nano-quercetin (N-QCT)-mediated aPDT against S. mutans. Also, in silico evaluation of the inhibitory effect of N-QCT on the competence-stimulating peptide (CSP) of S. mutans was performed to elucidate the impact of aPDT on various QS-regulated genes. METHODS: Cytotoxicity and intracellular reactive oxygen species (ROS) generation were assessed following synthesis and confirmation of N-QCT. Subsequently, the minimum biofilm inhibitory concentration (MBIC) of N-QCT against S. mutans and anti-biofilm effects of aPDT were assessed using colorimetric assay and plate counting. Molecular modeling and docking analysis were performed to confirm the connection of QCT to CSP. The metabolic activity of S. mutans and the expression level of various genes involved in QS were evaluated by flow cytometry and reverse transcription quantitative real-time PCR, respectively. RESULTS: Successful synthesis of non-toxic N-QCT was confirmed through several characterization tests. The MBIC value of N-QCT against S. mutans was 128 µg/mL. Similar to the crystal violet staining, the results log10 CFU/mL showed a significant degradation of preformed biofilms in the group treated with aPDT compared to the control group (P < 0.05). Following aPDT, metabolic activity of S. mutans also decreased by 85.7% (1/2 × MBIC of N-QCT) and 77.3% (1/4 × MBIC of N-QCT), as compared to the control values (P < 0.05). In silico analysis showed that the QCT molecule was located in the site formed by polypeptide helices of CSP. The relative expression levels of the virulence genes were significantly decreased in the presence of N-QCT-mediated aPDT (P < 0.05). CONCLUSIONS: The combination of N-QCT with blue laser as a QQ-strategy leads to maximum ROS generation, disrupts the microbial biofilm of S. mutans, reduces metabolic activity, and downregulates the expression of genes involved in the QS pathway by targeting genes of the QS signaling system of S. mutans.