Dioscorea bulbifera Mediated Synthesis of Novel Au_coreAg_shell Nanoparticles with Potent Antibiofilm and Antileishmanial Activity

2.1. Synthesis of Gold Core Silver Shell Nanoparticles (Au_coreAg_shellNPs)

DBTE was prepared as per our earlier report [7]. In brief, fresh tubers of D. bulbifera were washed thoroughly and chopped into slices and shade dried followed by grinding into fine powder. 5 g of the powder was boiled in 100 mL of distilled water for 5 min and decanted followed by filtration. Synthesis was initiated by addition of 5 mL of DBTE to 95 mL of aqueous solution with 1 mM HAuCl₄ and 0.7 mM of AgNO₃ followed by incubation at 50°C for 5 h. The progress of reduction was monitored by measuring the UV-visible spectra of the solution at regular intervals on a spectrophotometer (SpectraMax M5, Molecular Devices Corp, USA) operated at resolution of 1 nm. Bioreduced Au_coreAg_shellNPs synthesized by DBTE were centrifuged at 10,000 rpm for 15 min at room temperature, followed by redispersal of the pellet in sterile distilled water to remove any uncoordinated biological molecules. Repetition of alternate centrifugation and redispersion in sterile distilled water for three times ensured better separation of free entities from the nanoparticles which were used for all further biological activities.

2.2. Transmission Electron Microscopy (TEM), High Resolution Transmission Electron Microscopy (HRTEM), and Dynamic Light Scattering (DLS) Measurements

Shape and size of the bioreduced Au_coreAg_shellNPs were determined using TEM (Tecnai 12 cryo TEM, FEI, Netherland) and was confirmed by JEOL-JEM-2100 higher resolution transmission electron microscope (HRTEM) coupled with elemental composition mapping under scanning transmission electron microscopic mode (STEM). Energy dispersive spectra of Au_coreAg_shellNPs, recorded in the energy dispersive spectrometer (EDS) equipped in JEOL JSM 6360A analytical scanning electron microscope at an energy range 0–20 keV confirmed the synthesis of Au_coreAg_shellNPs. Particle size was analyzed using the dynamic light scattering equipment (Zetasizer Nano-2590, Malvern Instruments Ltd, Worcestershire, UK) in polystyrene cuvette.

2.3. Antibiofilm Activity

5 μL, OD₆₀₀ (0.05) adjusted overnight grown cultures of A. baumannii, P. aeruginosa, E. coli, and S. aureus were incubated in absence and in presence of Au_coreAg_shellNPs at various concentrations ranging from 1 to 1024 μg/well in Mueller Hinton broth in 96 well microtitre plates. Negative controls (no cells) were included for each of the experimental preparations. The microtitre plates were then incubated overnight at 37°C under static conditions and processed thereafter. Nonadherent cells were removed from microtitre by aspiration. Wells containing biofilm matrices were washed thrice with sterile PBS followed by staining with 0.1% gentian violet (HiMedia, India) for 10 min at room temperature. Excess stain was removed by immersing in water trough followed by drying in laminar air flow. Finally, 200 μL of absolute ethanol was added to each well and shaken at 1020 rpm for 10 sec. Absorbance at 570 nm was recorded in a multiplate reader. Biofilm indices were calculated after normalizing with appropriate controls. All biofilm assays were repeated thrice.

2.4. Visualization of Biofilm Employing SEM and AFM

The biofilm inhibition was carried out on sterile grease free glass cover slips as per the above mentioned procedure with a final volume of 2 mL in 6 well plates. After 24 h, cover slips were washed with sterile PBS followed by fixation with glutaraldehyde which was further dehydrated sequentially using ethyl alcohol and analyzed using scanning electron microscope (SEM, JEOL JSM 6360A).

2.5. Atomic Force Microscopy

2.6. Antileishmanial Activity

Promastigotes of Leishmania donovani (MHOM/IN/83/Ag83) were cultured in Medium 199 (Sigma, St. Louis, MO) supplemented with 10% heat inactivated foetal bovine serum (FBS) (Sigma, St. Louis, MO) at 25°C. The parasites were maintained in vitro by subpassaging them every fifth day. Antileishmanial activity of Au_coreAg_shellNPs was investigated using MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Logarithmic phase L. donovani promastigotes (2 × 10⁵ parasites/mL) were seeded in a 96-well microtitre plate and treated with the nanoparticles followed by incubation at 25°C for 72 h.

2.7. SEM and AFM Analysis

72 h treated L. donovani promastigotes were adhered to poly-L-lysine coated cover slips and fixed with 4% paraformaldehyde for 15 min at room temperature which were subjected to SEM and AFM analysis as per the procedure described in the earlier section.

2.8. DNA Fragmentation Assay

The assay was carried as described in earlier report by Herrmann et al., 1994 with a slight modification [41]. Briefly, L. donovani promastigotes were treated with nanoparticles for 72 h. The pellets were treated with lysis buffer (1% NP-40 in 2 mM EDTA, 50 mM Tris-HCl, pH 7.5) followed by centrifugation at 1600 ×g for 5 min. The supernatant was brought to 1% SDS and treated with RNAase (5 μg/μL) for 2 h at 56°C. Proteinase K (2.5 μg/μL) treatment was given for 3 h at 37°C followed by precipitation of DNA with 1/2 volume 10 M ammonium acetate and 2.5 volume ethanol. The DNA was dissolved in TE buffer (Tris 10 mM, EDTA 1 mM, pH 8.0) and analyzed by agarose gel electrophoresis.

3.1. UV-Visible Spectroscopy

DBTE mediated reduction of silver and gold ions in aqueous solution was confirmed by the gradual change in color from orange red to dark reddish violet as a function of reaction time. UV-vis spectra recorded from 0 to 5 h revealed a distinct and clear increase in intensity of the gold surface plasmon band, while a well-defined band corresponding to AgNPs was not observed in case of Au_coreAg_shellNPs (Figure 1(a)). Although no significant synthesis was observed from 0 to 0.5 h, but thereafter the rate of reduction took a rapid pace, almost completing the reduction within 4 h, with a slight increase in the intensity till 5 h. UV-vis spectra confirmed the synthesis of Au_coreAg_shellNPs which showed a peak at 540 nm unlike that of individual AgNPs at 440 nm (Figure 1(b)). However, a decrease in the peak intensity was observed in case Au_coreAg_shellNPs as compared to that of individual AuNPs at 540 nm which might be attributed due to formation of the silver shell onto a gold core.

3.2. TEM, HRTEM, and EDS Analysis

TEM images confirmed the size, shape, and nature of bioreduced nanoparticles. TEM images distinguished the shell from the core with prominent boundary between the two layers (Figure 2(a)). The diameter of the inner core was in a range between 5 and 11 nm while the entire particle showed a diameter of 13 to 19 nm. Particles were found to be almost monodispersed, the majority being spherical with a core represented by dark contrast surrounded by a shell with a lighter shade. Aggregation was not observed indicating the stable nature of the particles. HRTEM analysis was also in agreement with the TEM analysis where the diameter of the core and the entire particle was measured to be 6 to 10 nm and 13 to 18 nm, respectively (Figure 2(c)). Lattice fringes were clearly observed on the surface of the nanoparticles confirming the crystalline nature (Figure 2(c)). Additionally, elemental mapping clearly indicated the location of elemental silver and gold in the core shell nanoparticles (Figure 2(d)). It was distinctly evident that gold formed the core while silver formed the shell of the nanoparticles confirming the fact that the bioreduced nanoparticles were Au_coreAg_shell in nature. EDS results confirmed the presence of both elemental silver as well as elemental gold in the nanoparticles. Au_coreAg_shellNPs were comprised of 57.34 ± 1.01% gold and 42.66 ± 0.97% silver of the total mass (Figure 2(b)). Particle size analysis exhibited a variation mostly between 7 and 18 nm, the majority being 10 nm (Figure 2(a)).

3.3. Biofilm Inhibitory Activity

Biofilm inhibition studies revealed a high efficacy and selectivity of Au_coreAg_shellNPs towards controlling bacterial pathogens. Au_coreAg_shellNPs, at a concentration of 100 μg/mL, could inhibit biofilm formation up to 83.68 ± 0.09% against A. baumannii (Figure 3). However, Au_coreAg_shellNPs showed a comparatively lower activity equivalent to 18.93 ± 1.94% against P. aeruginosa at the same concentration. Biofilm formation in case of E. coli was inhibited up to 22.33 ± 0.56% which was found to be superior to P. aeruginosa while inferior as compared to A. baumannii. It is important to note that the biofilm formation was inhibited up to 30.70 ± 1.33% even against Gram positive bacteria like S. aureus.

3.4. Scanning Electron Microscopy for Biofilm Inhibition

Scanning electron micrographs revealed the effective inhibition of biofilm by Au_coreAg_shellNPs against A. baumannii. Untreated biofilm were represented by confluent lawns (Figure 4(a-i)) with intact cellular morphology. Treatment with Au_coreAg_shellNPs exhibited marked biofilm inhibition with discrete cells on the glass surface instead of lawns (Figure 4(a-ii)). The treated cells showed irregular morphology owing to cell damage by the Au_coreAg_shellNPs. A similar observation was achieved in case of P. aeruginosa as well. In case of the untreated cells a profound bacterial biofilm surrounded by exopolymeric slimy matrix was seen (Figure 4(b-i)). Treated cells exhibited biofilm inhibition characterized by prominent membrane disintegration leading to efflux of cellular materials (Figure 4(b-ii)). Size of E. coli cells were found to increase abruptly followed by efficient biofilm inhibition on treatment with Au_coreAg_shellNPs (Figure 4(c-ii)). Surface attachment was greatly compromised on treatment unlike the untreated cells which formed a compact biofilm due to effective adherence onto the glass surface (Figure 4(c-i)). In case of S. aureus, the untreated biofilm was characterized by groups of closely packed spherical cells (Figure 4(d-i)) which on treatment showed discontinuous biofilm consisting of sparsely packed cells in discrete aggregates. The cells in treated biofilm showed surface invaginations and deformities (Figure 4(d-ii)).

3.5. Atomic Force Microscopy

AFM images were recorded to confirm the effect of Au_coreAg_shellNPs treatment on bacterial biofilms which illustrated a packed lawn in case of untreated biofilms. The interrupted biofilms without packed morphology owing to compromised cell adhering capability in treated biofilm (Figure 5(a-ii)) were readily differentiated from the untreated bacterial biofilms (Figure 5(a-i)). A. baumannii showed complete elimination of biofilm characterized by discrete single cells attaching onto the glass surface. The measurements of elastic moduli showed that the treated cells (104.59 KPa) were stiffer as compared to untreated cells (0.82 KPa). P. aeruginosa biofilm exhibited cells embedded in the exopolymeric matrix while in case of treated biofilm, discrete cells were observed (Figures 5(b-i) and 5(b-ii)). Similar trend in the increase of stiffness in the treated cells (75.72 KPa) was observed compared to untreated cells (40.02 KPa). Untreated E. coli biofilm was characterized by evenly distributed uninterrupted confluent bacterial lawns while biofilms treated with Au_coreAg_shellNPs showed uneven cellular distribution with discrete solitary cells on the glass surface at large distances (Figures 5(c-i) and 5(c-ii)). Stiffness (57.64 KPa) in the treated biofilm increased as compared to untreated biofilm (22.84 KPa). Inhibition of S. aureus biofilms was clearly evident from the AFM micrographs showing the irregularly clumped cellular masses separated by large valleys unlike the untreated biofilm which exhibited profound uniform lawn (Figures 5(d-i) and 5(d-ii)). Stiffness in treated biofilm increased up to 197.45 KPa which was significantly higher compared to 48.63 KPa found for the untreated biofilm.

3.6. Antileishmanial Activity

The effect of Au_coreAg_shellNPs on L. donovani promastigotes was determined quantitatively by MTT. The parasites were killed effectively on treatment with Au_coreAg_shellNPs (MIC = 32 μg/mL). SEM micrographs of untreated cells showed intact spindle shaped morphology tapering at both ends with a long flagellum which is typical for L. donovani healthy promastigote (Figure 6(a)). However, cells treated with Au_coreAg_shellNPs showed a pronounced deformation characterized by complete loss of spindle shaped morphology finally becoming spherical (Figure 6(b)). Similar results were also obtained in case of AFM micrographs (Figures 6(c) and 6(d)) which confirmed the antileishmanial potential that were further supported by DNA fragmentation (Figure 6(e)).