Biosynthesized Silver Nanoparticle (AgNP) From Pandanus odorifer Leaf Extract Exhibits Anti-metastasis and Anti-biofilm Potentials

Abstract

Cancer and the associated secondary bacterial infections are leading cause of mortality, due to the paucity of effective drugs. Here, we have synthesized silver nanoparticles (AgNPs) from organic resource and confirmed their anti-cancer and anti-microbial potentials. Microwave irradiation method was employed to synthesize AgNPs using Pandanus odorifer leaf extract. Anti-cancer potential of AgNPs was evaluated by scratch assay on the monolayer of rat basophilic leukemia (RBL) cells, indicating that the synthesized AgNPs inhibit the migration of RBL cells. The synthesized AgNPs showed MIC value of 4-16 μg/mL against both Gram +ve and Gram -ve bacterial strains, exhibiting the anti-microbial potential. Biofilm inhibition was recorded at sub-MIC values against Gram +ve and Gram -ve bacterial strains. Violacein and alginate productions were reduced by 89.6 and 75.6%, respectively at 4 and 8 μg/mL of AgNPs, suggesting anti-quorum sensing activity. Exopolysaccharide production was decreased by 61-79 and 84% for Gram -ve and Gram +ve pathogens respectively. Flagellar driven swarming mobility was also reduced significantly. Furthermore, In vivo study confirmed their tolerability in mice, indicating their clinical perspective. Collective, we claim that the synthesized AgNPs have anti-metastasis as well as anti-microbial activities. Hence, this can be further tested for therapeutic options to treat cancer and secondary bacterial infections.

Keywords: anti-biofilm; anti-metastasis; molecular docking; quorum sensing; silver nanoparticles (AgNPs).

FIGURE 1

Characterization of POLE-capped silver nanoparticles (AgNPs). (A) Different concentrations of AgNO₃ (2, 4, 6, 8, and 10 mM) were used to synthesize the AgNPs. 5 mL of each leaf extract (50 mg/mL) and AgNO₃ solution (8 mM), gave best yield of silver nanoparticles by using microwave irradiation method. The resulting solution turned out to the characteristic brown color of the AgNPs in 90 min. (B) The absorption spectra of the synthesized nanoparticles were recorded in 300–800 nm wavelength range. The characteristic absorption peak of AgNPs is centered on 430 ± 20 nm. (C) The XRD data show diffraction peaks at 2θ = 37.2, 43.4°, 63.5, and 76.6° corresponding to the [111], [200], [220], and [311] planes of silver, respectively. (D) FT-IR spectra of plant extract (POLE) and AgNPs. The characteristic peak at 1636 cm^-1 can be attributed to the C = O (carbonyl) groups. (E) Surface enhanced Raman spectroscopy (SERS), spectra displayed the characteristic signals of POLE with the enhanced intensity for AgNPs indicating the capping of POLE on the surface of AgNPs. (F) TEM image of POLE-capped AgNPs, (G,H) HRTEM (high-resolution transmission electron microscopy) images of POLE-capped AgNPs, clearly demonstrated the stability of the particles even after a period of 4 months.

FIGURE 2

Cellular toxicity and cell migration in response of AgNPs. (A) Cytotoxicity of AgNPs on RBL cells were determined by MTT assay. MTT salt gets reduced by reducing enzymes into water-insoluble formazan. The images of the cells with AgNPs showed the dosage dependent effect of the AgNPs (1–10 μg/mL). (B) Determination of IC₅₀ value (estimated to be 3.40 μg/mL) of AgNPs on RBL cells (data represented as SEM). (C) Scratch was made onto a monolayer of RBL cells and treated by 3 μg/mL of AgNPs (denoted as T, while the control as C) for different time points. Comparison of migration in both C and T was made by taking images at different time intervals (0, 24, 48, and 72 h) [control -(i), (ii), (iii), (iv) and test -(v), (vi), (vii), (viii)]. Results represented by marking the scratch with parallel lines and visually displaying the number of cells migrated in to the scratch area. The scale bar in (A,C) represents 100 μm.

FIGURE 3

Anti-quorum sensing (QS) and anti-biofilm properties of AgNPs. (A) To test the effect of synthesized nanoparticles during the process of quorum sensing, the violacein was quantified by taking the OD at 585 nm in different conditions. Different concentrations of AgNPs showed the significant dosage dependent manner reduction in violacein production by Chromobacterium violaceum. Data showed the QS-mediated inhibition of violacein by AgNPs. (B) The inhibition of biofilm formation assessed by estimating the exopolysaccharide (EPS) production in presence and or absence of different concentrations of AgNPs was performed. Data showed the reduction in EPS production with increasing AgNPs concentration. (C) The reduced alginate production in response to different concentrations of AgNPs has been quantified. The presence of synthesized AgNPs showed 26.3–75.6% reduction in alginate production by Pseudomonas aeruginosa. (D) The biofilm formation was assessed directly by taking OD at 470 nm in microtiter plate. AgNPs presence causes the reduction of biofilm biomass production in the range of 22–79, 29–87, 12–59, 22–63, and 17–81% by P. aeruginosa, E. coli, C. violaceum, K. pneumoniae, and S. aureus respectively. One-way ANOVA with Dunnet post-test for (A,C), in comparison to zero dosage of AgNPs, while one-way ANOVA with multiple comparison applied in (B,D). Means denoted by the same letter within parameter are not significantly different at p ≤ 0.05, using DMRT.

FIGURE 4

Inhibition of biofilm of E. coli, P. aeruginosa, C. violaceum, K. pneumoniae, and S. aureus by AgNPs under scanning electron microscope (SEM). Biofilm formation by different bacterial agents were assessed by SEM. The biofilm treated by ½ × MIC of AgNPs showed reduction in the biofilm mass production compared with control untreated (scale bar 10 μm).

FIGURE 5

In vivo effect of AgNPs on the toxicity of liver and kidney enzymes. Effect of AgNPs on liver and kidney enzymes were assessed to determine, whether synthesized nanoparticle has any cellular toxicity. Data showed (A) reduced alanine aminotransferase (ALT) activity, (B) aspartate aminotransferase (AST) activity, (C) urea level, (D) creatinine level in response to the treatment of nanoparticles. (E) Comet assay performed to see the effect of synthesized nanoparticles in nuclear-DNA damage. The liver cells treated showed a decrease in the length of DNA damage tail by 32.84 and 23.95% at two different dosage (1 and 2 mg/kg respectively) with respect to positive control. (F) Kidney cells, also showed the same effect of the reduction (31.75 and 20.34%) at two different dosage (1 and 2 mg/kg respectively) in olive tail movement indicative of DNA damage (P < 0.05, one-way ANOVA with Dunnet post-test in comparison between positive control and nanoparticle treatments). ^∗∗∗indicate the significant differences in comparison to negative control (CN-), with p vale < 0.005. ^###indicate the significant differences in comparison to positive control (CN+), with p vale < 0.005; ^##indicates significantly different from control positive (CN+) at p < 0.05.

FIGURE 6

Synthesized AgNPs inhibit DNA damage. Representative images of the comet assay to estimate the DNA damage in cells treated with nanoparticles. Different groups were assigned as negative control (CN–), positive control (CN+), nanoparticle treated; dosage 1 mg/kg (NP-1), and nanoparticle treated; dosage 2 mg/kg (NP-2) in liver and kidney cells.

FIGURE 7

Molecular docking determine the AgNP’s binidng capacity to various enzymes. Molecular docking was performed to idnetify the interaction of synthesized nanoparticles with amino acid residues. The docking model show an interaction of Ag to LasR through Val83 and Vfr through Tyr65, Asp127, and Lys131. further RhlR interacted with Ag through Ile124, Ala126, Pro127, Glu160, Thr163, and Gln164. Similarly the docking of Ag with (A) LasR, (B) Vfr, (C) QscR, (D) RhlR, and (E) PqsA.

FIGURE 8

Model showing the systematic approch for the synthesis of nanoparticle (AgNPs) and their characterization. Three stages of the model showing the basic steps for the synthesis of silver nanoparticles (AgNPs), their characterization to confirm the nature of nanoparticles and biologcal functions. As the AgNPs have anti-biofilm and anti-cancer propertise, the synthesized AgNPs could be further tested for many therapeutics options and clinical managements.