In-Vitro Antibacterial and Anti-Inflammatory Effects of Surfactin-Loaded Nanoparticles for Periodontitis Treatment

Abstract

Periodontitis is an inflammatory disease associated with biofilm formation and gingival recession. The practice of nanotechnology in the clinical field is increased overtime due to its potential advantages in drug delivery applications. Nanoparticles can deliver drugs into the targeted area with high efficiency and cause less damages to the tissues. In this study, we investigated the antibacterial and anti-inflammatory properties of surfactin-loaded κ-carrageenan oligosaccharides linked cellulose nanofibers (CO-CNF) nanoparticles. Three types of surfactin-loaded nanoparticles were prepared based on the increasing concentration of surfactin such as 50SNPs (50 mg surfactin-loaded CO-CNF nanoparticles), 100SNPs (100 mg surfactin-loaded CO-CNF nanoparticles), and 200SNPs (200 mg surfactin-loaded CO-CNF nanoparticles). The results showed that the nanoparticles inhibited the growth of Fusobacterium nucleatum and Pseudomonas aeruginosa. The reduction in biofilm formation and metabolic activity of the bacteria were confirmed by crystal violet and MTT assay, respectively. Besides, an increase in oxidative stress was also observed in bacteria. Furthermore, anti-inflammatory effects of surfactin-loaded CO-CNF nanoparticles was observed in lipopolysaccharide (LPS)-stimulated human gingival fibroblast (HGF) cells. A decrease in the production of reactive oxygen species (ROS), transcription factor, and cytokines were observed in the presence of nanoparticles. Collectively, these observations supported the use of surfactin-loaded CO-CNF as a potential candidate for periodontitis management.

Keywords: anti-inflammatory; antibacterial; nanoparticle; periodontitis; surfactin.

Figure 1

Schematic representation of surfactin-loaded nanoparticles preparation.

Figure 2

(A) Determination of antimicrobial properties of nanoparticles against (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa. (B) Zone of inhibition of nanoparticles. Data are expressed as mean ± SD (n = 3). The asterisk (*) represents a significant difference (p < 0.05) from the positive control (doxycycline) analyzed by Tukey’s test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200S NPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 3

(A)Visualization of biofilm formation by (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa and (B) quantification of biofilm inhibition in Fusobacterium nucleatum and Pseudomonas aeruginosa. Data are expressed as mean ± S.D. (n = 3). The asterisk (*) represent a significant difference (p < 0.05) from positive control (doxycycline) analyzed by Tukey’s test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 4

Effects of nanoparticles on the metabolic activity of Fusobacterium nucleatum and Pseudomonas aeruginosa. Data are expressed as mean ± S.D. (n = 3). The asterisk (*) represent significant differences (p < 0.05) from positive control (doxycycline) analyzed by Tukey’s test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200S NPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 5

Effects of nanoparticles on the malondialdehyde level of (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa. Data are expressed as mean ± S.D. (n = 3). The values with different letters (a–c) represent significantly different (p < 0.05) as analyzed by Tukey’s test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 5

Effects of nanoparticles on the malondialdehyde level of (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa. Data are expressed as mean ± S.D. (n = 3). The values with different letters (a–c) represent significantly different (p < 0.05) as analyzed by Tukey’s test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 6

Acridine orange (AO) assay of (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa treated with different samples. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 6

Acridine orange (AO) assay of (a) Fusobacterium nucleatum and (b) Pseudomonas aeruginosa treated with different samples. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 7

Cell viability of human gingival fibroblast cells (1 × 10⁴ cells/well) in the presence of nanoparticles after 24 h. Data are expressed as mean ± S.D. (n = 3). The asterisk (*) indicated the significant differences of the groups from control at p < 0.05 analyzed by the Tukey test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 8

(a) nitric oxide production, (b) inhibition of nitroblue tetrazolium reduction, and (c) malondialdehyde level in nanoparticles treated lipopolysaccharide (LPS) stimulated human gingival fibroblast cells. The cells (1 × 10⁴ cells/well) were pre-treated with 20 µL of samples (50 µg/mL) and 10 µL of LPS (1 µg/mL). Data are expressed as mean ± S.D. (n = 3). The letters (a–c) indicated the significant differences of the groups at p < 0.05 analyzed by the Tukey test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 8

(a) nitric oxide production, (b) inhibition of nitroblue tetrazolium reduction, and (c) malondialdehyde level in nanoparticles treated lipopolysaccharide (LPS) stimulated human gingival fibroblast cells. The cells (1 × 10⁴ cells/well) were pre-treated with 20 µL of samples (50 µg/mL) and 10 µL of LPS (1 µg/mL). Data are expressed as mean ± S.D. (n = 3). The letters (a–c) indicated the significant differences of the groups at p < 0.05 analyzed by the Tukey test. CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.

Figure 9

Level of (a) nuclear factor (NF)-κB, (b) prostaglandin E2, and (c) interleukin-6 in nanoparticle treated lipopolysaccharide (LPS)-stimulated cells. The cells (1 × 10⁴ cells/well) were pre-treated with 20 µL of samples (50 µg/mL) and 10 µL of LPS (1 µg/mL). Data are expressed as mean ± S.D. (n = 3). The letters (a–c) indicated the significant differences of the groups at p < 0.05 analyzed by the Tukey test. Abbreviations: CO-CNF: κ-carrageenan oligosaccharides linked cellulose nanofibers; 50SNPs: 50 mg surfactin-loaded CO-CNF nanoparticles; 100SNPs: 100 mg surfactin-loaded CO-CNF nanoparticles; 200SNPs: 200 mg surfactin-loaded CO-CNF nanoparticles.