Non-thermal Atmospheric Pressure Plasma Combined with Benzoyl Peroxide to Enhance the Inhibition of Bacteria

Technical Summary: Non-Thermal Atmospheric Pressure Plasma Combined with Benzoyl Peroxide to Enhance Bacterial Inhibition

Problem Statement
While Cold Atmospheric Plasma (CAP) has emerged as a promising antibacterial agent due to its ability to generate Reactive Oxygen Species (ROS) without causing thermal damage, the study identifies a need to further enhance its bactericidal efficacy. Complete eradication of bacteria is critical to prevent treatment failure and secondary infections, particularly in complex biomedical structures like dentinal tubules. Although plasma generates hydroxyl radicals and peroxynitrite, the authors propose that combining plasma with chemical agents could offer a novel strategy to amplify antibacterial effects. Specifically, the study investigates whether integrating Benzoyl Peroxide (BPO)—a compound known for its oxidative antibacterial properties and ability to generate benzoyloxyl radicals upon decomposition—can synergize with plasma to improve bacterial inactivation.

Methodology
The research utilized a dielectric barrier discharge (DBD) plasma jet system. The setup consisted of an alumina tube (3 mm inner/5 mm outer diameter) with a stainless steel high-voltage electrode and an aluminum tape grounded electrode. The system operated with Argon gas as the working medium.

• Parameter Optimization: Optical Emission Spectroscopy (OES) was employed to determine optimal operating conditions. Researchers varied the power supply voltage (3–4.3 kV), argon flow rate (2–7 SLM), and oxygen mixing ratios (0–2.4%). Surface temperature was monitored using a Forward Looking Infrared (FLIR) camera on ceramic substrates to ensure thermal safety. UV emission was measured to assess potential photolytic effects on BPO.
• Chemical Analysis: A chemical titration method (using potassium iodide and sodium thiosulfate) was used to quantify BPO content before and after 5 minutes of plasma exposure to verify if plasma directly induced BPO decomposition.
• Antibacterial Assay: Escherichia coli cultures were spread on agar plates. The study compared the zones of inhibition for three conditions: plasma alone, plasma with 10 µL of BPO, and plasma with 16.7 µL of BPO. Treatments were conducted at a fixed distance of 17 mm for durations of 60, 90, and 120 seconds.

Key Results

• Plasma Characterization: OES analysis revealed that the highest emission intensity at 309 nm (indicative of OH radicals) occurred at 4.3 kV and an argon flow rate of 4 SLM. The addition of oxygen to the working gas did not significantly enhance ROS generation at low concentrations and caused a decrease in plasma density at concentrations above 2%; thus, pure argon was selected for subsequent experiments.
• Thermal and Optical Properties: After 5 minutes of exposure, the surface temperature of the ceramic substrate rose from 18.4°C to 27.3°C, confirming the non-thermal nature of the treatment. UV measurements indicated emission in the 236–300 nm and 365–480 nm ranges, but the estimated photon flux suggested that direct photolysis of BPO was likely limited under these conditions.
• Chemical Decomposition: Chemical titration showed no significant reduction in BPO content after 5 minutes of plasma treatment (79.1 mg vs. 78.7 mg in a 100 mg sample). The authors conclude that while plasma may not directly decompose BPO in bulk quantities under these conditions, it cannot be ruled out that transient radical interactions or re-formation of BPO masked the results.
• Antibacterial Efficacy: The combination of plasma and BPO demonstrated a significant synergistic effect. The zone of inhibition for E. coli increased with BPO addition. After 120 seconds of treatment, the inhibition zone grew from 0.91 ± 0.100 cm² (plasma alone) to 1.2 ± 0.092 cm² (with 10 µL BPO) and 2.21 ± 0.088 cm² (with 16.7 µL BPO).

Significance and Claims
The paper claims that combining non-thermal atmospheric pressure plasma with Benzoyl Peroxide significantly enhances bactericidal efficacy against E. coli compared to either treatment alone. The study suggests that the mechanism is likely not driven by direct photolytic decomposition of BPO by plasma UV, but rather by synergistic interactions. The authors propose several plausible mechanisms for this enhancement:

1. Plasma may disrupt bacterial envelopes, increasing susceptibility to BPO or facilitating its penetration.
2. The simultaneous action of plasma-generated ROS and BPO may drive bacteria to an irreversible state of damage more rapidly.
3. Interactions between plasma species and BPO may generate additional harmful substances.

The authors emphasize that while the precise chemical mechanisms remain to be fully elucidated, the observed expansion of inhibition zones supports the feasibility of this combined approach as a potential strategy for improving disinfection in dermatological and dental applications, such as root canal therapy and acne treatment, where deep penetration and complete bacterial eradication are required.