Attenuation of Aggregatibacter actinomycetemcomitans virulence using curcumin-decorated nanophytosomes-mediated photo-sonoantimicrobial chemotherapy

Abstract

This study aimed to focus on the simultaneous use of antimicrobial photodynamic therapy (aPDT) and sonodynamic antimicrobial chemotherapy (SACT), which is called photo-sonodynamic antimicrobial chemotherapy (PSACT) to attenuate the virulence of Aggregatibacter actinomycetemcomitans. Following the synthesis of Curcumin-decorated nanophytosomes (Cur-NPhs) as a novel photo-sonosensitizer, its particle size, polydispersity, ζ-potential surface morphology, physical stability, drug release, and entrapment efficiency were determined. In the Cur-NPhs-PSACT, the antimicrobial activities of Cur-NPhs against A. actinomycetemcomitans were investigated using cell viability, biofilm killing/degradation, metabolic activity, expression of quorum-sensing-associated qseB and qseC genes, and biofilm-associated rcpA gene under blue laser irradiation plus ultrasonic waves. Characterization tests showed the presence of a sphere-shaped vesicle and the self-closed structure of Cur-NPhs, resulting in a high drug-loading content and encapsulation efficiency. However, the antimicrobial effect of Cur-NPhs-PSACT was dose-dependent, PSACT using the high concentrations of Cur-NPhs (50 × 10^-4 g/L) showed significant reductions (P < 0.05) in cell viability (13.6 log₁₀ CFU/mL), biofilm killing/degradation (65%), metabolic activity (89.6%,), and mRNA levels of virulence determinant genes (qseB; 9.8-fold, qseC; 10.2-fold, and recA; 10.2-fold). This study concludes that the Cur-NPhs-PSACT had antimicrobial activities against A. actinomycetemcomitans by downregulating the expression of virulence genes, and may attenuate this bacterium that decreases periodontal disease severity in patients.

Figure 1

Characterization of synthesized Cur-NPhs. (a) Representative scanning electron micrograph (scale bar represents 10 μm). (b) Representative transmission electron micrograph (scale bar represents 30 nm). (c) Average particle size. (d) ζ-potential value.

Figure 2

Profile of Cur release from Cur-NPhs.

Figure 3

Particle size, polydispersity index, and ζ-potential of Cur-NPhs in different conditions; (a) at 4 °C, (b) at 37 °C.

Figure 4

Retention rate percentage of Cur-NPhs at different pH; (a) during 24 h and (b) 48 h storage period.

Figure 5

Cell viability of A. actinomycetemcomitans following different treatments. Significant differences according to the control, *P < 0.05.

Figure 6

Biofilm degradation ability of A. actinomycetemcomitans following different treatments. Significant differences according to the control, *P < 0.05.

Figure 7

Metabolic activity of A. actinomycetemcomitans following different treatments. Significant differences according to the control, *P < 0.05.

Figure 8

Specificity of the designed primers; a. A 2% agarose gel electrophoresis for detection of qseB, qseC, and 16S rRNA. L ladder 100 bp, i: qseB, ii: qseC, iii: 16S rRNA. b. Melting curve, i: qseB, ii: qseC, iii: 16S rRNA.

Figure 9

Gene expression levels of qseB and qseC following different treatments. Significant differences according to the control, *P < 0.05.

Figure 10

Gene expression levels of rcpA following different treatments. Significant differences according to the control, *P < 0.05.

Figure 11

Schematic apparatus for illumination treatment of sample.