Polyethyleneimine nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo

Abstract

Incorporation of cross-linked quaternary ammonium polyethylenimine (QPEI) nanoparticles in dental resin composite has a long-lasting and wide antimicrobial effect with no measured impact on biocompatibility in vitro. We hypothesized that QPEI nanoparticles incorporated into a resin composite have a potent antibacterial effect in vivo and that this stress condition triggers a suicide module in the bacterial biofilm. Ten volunteers wore a removable acrylic appliance, in which two control resin composite specimens and two resin composite specimens incorporating 1% wt/wt QPEI nanoparticles were inserted to allow the buildup of intraoral biofilms. After 4 h, the specimens were removed and tested for bacterial vitality and biofilm thickness, using confocal laser scanning microscopy. The vitality rate in specimens incorporating QPEI was reduced by > 50% (p < 0.00001), whereas biofilm thickness was increased (p < 0.05). The ability of the biofilm supernatant to restore bacterial death was tested in vitro. The in vitro tests showed a 70% decrease in viable bacteria (p < 0.05). Biofilm morphological differences were also observed in the scanning electron microscope micrographs of the resin composite versus the resin composite incorporating QPEI. These results strongly suggest that QPEI nanoparticles incorporated at a low concentration in resin composite exert a significant in vivo antibiofilm activity and exhibit a potent broad spectrum antibacterial activity against salivary bacteria.

Fig. 1.

Removable acrylic appliance withholding two resin composite discs and two discs of resin composite incorporating QPEI. Confocal laser microscope surface images of the attached biofilm formed on (1) resin composite mainly show dead cells stained red, whereas biofilm formed on (2) resin composite incorporating QPEI nanoparticles mainly shows live cells stained green.

Fig. 2.

Biofilms formed on resin composite incorporating QPEI nanoparticles and nonmodified resin composite. Confocal laser scanning microscope cross-section images of biofilms formed on resin composite (A) and resin composite with incorporated QPEI nanoparticles (B).

Fig. 3.

Bacterial vitality and biofilm thickness of biofilms formed on resin composite incorporating QPEI nanoparticles and nonmodified resin composite. Biofilms were stained using the BacLight LIVE/DEAD viability stain and scanned using a confocal laser scanning microscope. Average cross-section enumeration of viable and nonviable bacteria, as measured by Image ProPlus softwear of biofilms formed on resin composite (A) and resin composite with incorporated QPEI nanoparticles (B).

Fig. 4.

Biofilms formed on resin composite incorporating QPEI nanoparticles and on nonmodified resin composite. Scanning electron microscope micrographs (10,000×) of biofilms formed on resin composite (A) and resin composite with incorporated QPEI nanoparticles (B).

Fig. 5.

In vitro antibacterial effect of (i) resin composite with biofilm extract; (ii) resin composite incorporating 1% wt/wt of added QPEI nanoparticles, and (iii) nonmodified resin composite and (iv) microtiter plate surface on salivary bacteria. Kinetic measurements depicting growth of bacteria collected from saliva following direct contact with resin composite; resin composite incorporating 1% wt/wt of added QPEI nanoparticles; and with resin composite covered with an extract of bacteria previously grown on resin composite incorporating QPEI. Growth of the shed bacteria from the biofilm was measured every 20 min for 16 h. Each point on the curve is the average (± SD) optical density (650 nm) measured in 12 replica wells similarly prepared in the same microtiter plate. The linear portion of the logarithmic growth phase was analyzed and expressed according to two variables: the slope (a) and the constant (b) of the linear function ax + b = y. The slope (a) and the constant (b) correlate with growth rate and initial bacterial number, respectively.