Green silver nanoparticles for drug transport, bioactivities and a bacterium (Bacillus subtilis)-mediated comparative nano-patterning feature

Abstract

The ‘green’ synthetic aspects of functionally potent and biologically relevant nanomaterials are a crucial research objective. Pursuing this concept, we have investigated a green synthetic scheme for the sunlight-mediated generation of luminescent silver nanoparticles, which become stabilized via a supramolecular hydrogel (SHGel) network, as already reported by our group. In vitro and in vivo toxicity studies confirm the biologically relevant nature of SHGel-capped Ag NPs. Nontoxic SHGel-capped Ag NPs were intelligently used for the transport of drugs, including antifilarial and antibiotic agents, into cells. Apart from this activity, SHGel-capped Ag NPs and our previously reported nontoxic DNA hydrogel-capped Ag NPs are potent against pathogens and parasites. Most interestingly, the nanostructural patterns of SHGel- and DNA hydrogel-capped Ag NPs have been transformed into cotyledon- and flower bud-shaped forms of nanosilver, respectively, during their chemotherapeutic action against a particular bacterium, Bacillus subtilis. Transmission electron microscopy was used for the visualization of several patterns of nanosilver and the incorporation of Ag NPs into macrophages.

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1. Introduction

Nanodimensional phenomena of matter are an attractive aspect of science owing to their employment in modern life.¹ Nanomaterials are significant for their numerous applications including gas separation,² carbon nanotubes,³ nanoelectronics,⁴ etc. Nanomaterials are valued not only in materials science but also in medical science for their multifaceted uses⁵ such as tissue engineering,⁶ drug delivery,⁷ hyperthermia therapy,⁸ chemotherapy,⁹ etc.

Metallic nanoparticles, in particular gold and silver nanostructures, are intriguing because they exhibit great ability in terms of numerous functionalities¹⁰ such as food storage safety,¹¹ paints,¹² water purification¹³ and even food supplements.¹⁴ The connectivity of nanomaterials with biomolecules has appeared as a combined field of biology and chemistry.¹⁵ Among the different metallic nanoparticles, Ag NPs are distinct for their unique physicochemical properties along with remarkable bioactivities.¹⁶ Owing to their larger surface-to-mass ratio than that of bulk silver, Ag NPs have an increased ability to adsorb and carry drugs, probes, proteins, etc.¹⁷ Currently, Ag NPs are even being considered as antibiotics in the place of commonly used antibiotics because of their incredible antibacterial power.¹⁸ Owing to their remarkable fluorescent properties, Ag NPs are widely applicable in the fields of biology, medical imaging, chemical signalling, etc.^19–23 To obtain optimal biologically relevant therapeutic agents, a ‘green’ synthetic strategy for Ag NPs has been adopted.^24–26

Based on recorded databases, we are trying to provide a green synthetic approach to Ag NPs. We have photochemically synthesized luminescent Ag NPs and studied their biologically relevant nature. In this work, we have used our previously reported luminous supramolecular hydrogels (i.e., SHGel)²⁴ as a potential capping agent for the sunlight-directed photochemical synthesis of Ag NPs. This sunlight-mediated synthetic strategy for SHGel-capped Ag NPs is consistent with the principles of green chemistry.²⁶ Besides, owing to their nontoxic and biologically relevant features SHGel-capped Ag NPs are also investigated for cellular drug transport.

Recently, we have reported a sunlight-mediated formation strategy for DNA hydrogel-capped Ag-NPs.²⁴ DNA hydrogel-capped Ag NPs²⁴ are also more biocompatible and extremely nontoxic due to the presence of DNA hydrogel²⁴ (i.e., a calf thymus DNA-based hydrogel network) as a nanoparticle-stabilizing agent. In this work we have tried to differentiate the bioactivity of these diverse biofriendly silver nanoparticles, including DNA hydrogel-capped Ag-NPs²⁴ and SHGel-capped Ag NPs, towards different biosystems. The biofunctionality of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs²⁴ has been investigated against pathogens and parasites such as fungi (Pichia guilliermondii), bacteria (Escherichia coli DH5α and Bacillus subtilis) and microfilariae (Setaria cervi). Most interestingly, two different patterns of nanosilver, i.e., cotyledon- and flower bud-shaped Ag NPs have been created during the chemotherapeutic action of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs, respectively, against the bacterium Bacillus subtilis. It was experimentally found that in terms of bioactivities in the abovementioned cases DNA hydrogel-capped Ag NPs are more effective than SHGel-capped Ag NPs.

2. Experimental section

2.1 Materials

Different chemical species and associated consumable items were purchased from Sigma-Aldrich, Merck, Thermo Scientific, and NEST Biotechnology and utilized as received. Nuclease-free type 18 Milli-Q water was used in this work. Calf thymus DNA (i.e., CT-DNA), which was purchased from Merck, was employed as the source of DNA throughout the experiments.

2.2 Characterization

Absorption and fluorescence spectral analyses were performed using a Shimadzu UV-3101PC spectrophotometer and a PerkinElmer LS55 fluorescence spectrometer, respectively. Solid-state infrared spectroscopy was carried out on a Shimadzu FTIR-8400S spectrometer between 400 and 4000 cm⁻¹ using the KBr pellet method. Circular dichroism spectroscopic data were recorded using an Applied Photophysics Chirascan CD with a detection range of 160–850 nm. TEM microstructural images were recorded with a FEI Tecnai G² F30 S-Twin microscope utilizing an accelerating voltage of 300 kV. The TEM was also equipped with a Gatan Orius CCD camera in high-angle annular dark-field scanning transmission electron microscopy (STEM-HAADF) mode with a HAADF detector from Fischione (Model 3000). Compositional analysis was carried out by an energy-dispersive X-ray spectroscopy (EDS, EDAX, Inc.) attachment on the Tecnai G² F30. Energy-filtered TEM (EFTEM) measurements were performed using a Gatan Imaging Filter (Quantum SE model 963). The samples were dispersed in deionized water by sonication and dropped onto a conventional carbon-coated copper grid. A UV microscopy study was performed with a Leica 224 fluorescence microscope with a UV attachment. Fluorescence microscopy studies were carried out using an inverted fluorescence microscope (Victory Dewinter, Italy). Cell cultures were performed in a humidified CO₂ incubator (New Brunswick, Eppendorf, Germany). Dynamic light scattering (DLS) studies and measurements of the zeta potential distribution were performed with a Malvern instrument.

2.3 Ethical clearance

Animal-based investigations were permitted by the Institutional Ethical Committee for Animals and Humans, Visva-Bharati, India.

2.4 Synthesis of luminescent SHGel-capped Ag NPs

SHGel²⁴ (1.10 g) and solid silver nitrate (0.16 g) were mixed on a glass plate and then the mixture was subjected to open exposure to direct sunlight for ∼40 minutes, leading to the transformation into a pinkish-violet coloured mass from a white colour, which indicated the reduction of silver ions (Ag⁺) to silver nanoclusters (Ag⁰).^24,27 The microstructure of the pinkish-violet mass was examined using transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS) and energy-filtered TEM (EFTEM) techniques. The behaviour of SHGel-capped Ag NPs in an aqueous medium was also investigated by DLS studies and measurements of the zeta potential distribution. The fluorescence property of Ag NPs was tested by fluorescence spectroscopy and microscopic analyses.

2.5 Preparation of SHGel-capped Ag NPs and drug composites

SHGel-capped Ag NPs were mixed with four different drugs, namely, streptomycin (a broad-spectrum antibacterial antibiotic), albendazole (a broad-spectrum anthelmintic), ivermectin (an effective insecticide and antiparasitic drug) and diethylcarbamazine (an effective antifilarial drug) to obtain solid nanodrug composites by nanoprecipitation followed by lyophilization at −40 °C. Drug solutions were prepared in water (for streptomycin only) and 1% dimethyl sulfoxide solution (for the remaining three drugs) at a specific concentration of 100 μg mL⁻¹ individually and added dropwise to an aqueous solution of SHGel-capped Ag NPs of equal strength ([Ag NPs] = 100 μg mL⁻¹) under continuous stirring at 700 rpm for 6 h at ambient temperature and atmospheric pressure. Nanodrug composites were formed progressively, as observed from the appearance of a turbid suspension. The solvent was removed by lyophilization at −40 °C until dry. The formation of Ag NPs and drug composites was determined by FTIR spectroscopy.

2.6 Cellular incorporation of SHGel-capped Ag NPs

Isolated peritoneal macrophages (2 × 10⁶) from adult Wistar rats (160 ± 20 g) were cultured in vitro in six-well culture plates with RPMI-1640 medium supplemented with 100 μg mL⁻¹ streptomycin, 100 U penicillin and 100 μg mL⁻¹ amphotericin B. Aqueous solutions of luminescent SHGel-capped Ag NPs at different concentrations (i.e., 2.5, 5 and 10 μg mL⁻¹) were aseptically added to each culture medium and subsequently the culture plates containing the two vital ingredients of cells and Ag NPs were also incubated for 6 h at 37 °C with an atmosphere of 5% CO₂ in a humidified incubator. The treated cells were isolated by trypsinization and thoroughly washed with cold 100 mM PBS solution. Finally, the cells were fixed by glutaraldehyde to visualize the treated cells by TEM analysis following a literature method.²⁸

2.7 In vitro cytotoxicity evaluation of SHGel-capped Ag NPs

For in vitro cytotoxicity evaluation, a group of aqueous solutions of different doses (namely, 5, 10 and 25 μg mL⁻¹) of SHGel-capped Ag NPs were aseptically added to rat peritoneal macrophages (2 × 10⁶) plated in six-well culture vessels, which were incubated for 6 h and, after incubation, 200 μL MTT solution (in 100 mM PBS) was added to each of these culture vessels with incubation for a further 1 hour at 37 °C in a dark environment. Finally, the treated cells were isolated by scraping and centrifuged at 4000 rpm for 2 min. A total of 200 μL DMSO was added to each of the treated solutions containing cells and the colour intensities were determined at 495 nm using a plate reader (Beckman, USA) in each case.

2.8 In vivo toxicity evaluation of SHGel-capped Ag NPs

Separate groups of adult male Wistar rats were subjected to chronic exposure to SHGel-capped Ag NPs (100 μg) with one control group (n = 6; 120 ± 10 g) intraperitoneally, subcutaneously and orally for 30 days consecutively. All the rats were supplied with water and fed ad libitum with constant monitoring for any behavioural abnormalities and/or weight loss. Various serological and biochemical parameters were evaluated using conventional standard protocols.

N. B.: The biocompatible nature of DNA hydrogel-capped Ag NPs has already been reported by our group.²⁴

2.9 Circular dichroism spectroscopic study of SHGel-capped Ag NPs with calf thymus DNA

Aqueous solutions of calf thymus DNA (with a concentration of 1 μg mL⁻¹) and SHGel-capped Ag NPs (with a concentration of 1 μg mL⁻¹) were used for this study. The instrumental parameters were set at a scanning speed of 50 nm min⁻¹, a bandwidth of 1.0 nm and a sensitivity of 100 millidegrees. Four scans were averaged and smoothed to improve the signal-to-noise ratio. Values of molar ellipticity were expressed in terms of mean residue ellipticity (in deg cm² dmol⁻¹). Secondary structural analysis was performed using software supplied with the Applied Photophysics Chirascan CD.

2.10 Assessment of bioactivity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs²⁴

The bioactivities of both synthesized silver nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs²⁴) were tested against a filarial parasite (Setaria cervi), a pathogenic fungus (Pichia guilliermondii) and bacteria (Escherichia coli as a Gram −ve form and Bacillus subtilis as a Gram +ve form).

2.10.1 Antifilarial activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag-NPs. Setaria cervi (macro- and microfilariae) was cultured in RPMI-1640 medium supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U mL⁻¹ streptomycin, 100 μg mL⁻¹ penicillin, 0.25 μg mL⁻¹ amphotericin B, and 10% FBS in sterile culture plates containing 10 mL medium for macrofilariae (one male and one female, or two females) or 2 mL for microfilariae (n = 1.0 × 10⁴) with SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs in varying concentrations. The extent of parasite mortality was determined by repeated MTT assays.²⁹ The LC₅₀ values were calculated from the results of the MTT assays. The death of the parasites might be achieved via apoptosis, as found by acridine orange/ethidium bromide staining in fluorescence microscopic imaging.

2.10.2 Determination of antifungal activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs. The in vitro antifungal activities of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs were measured against the pathogenic fungal strain Pichia guilliermondii, which was formerly known as Candida guilliermondii (Genbank accession no. KC771883).³⁰ Fungal cultures on YPD agar medium (2% peptone, 1% yeast extract, 2% dextrose and 6% FBS) were treated with filter paper discs with a range of concentrations of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs for 24 h at 37 °C. The antifungal activity of these silver nanoparticles was determined by measuring the zone of inhibition that appeared around the paper disc on the culture plate following a literature method.³⁰

2.10.3 Analysis of antibacterial activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs²⁴. To investigate the in vitro antibacterial activity of the two synthesized nanoparticles, filter paper discs (Whatman no. 1) containing indicated concentrations of both SHGel-capped and DNA hydrogel-capped Ag NPs²⁴ dissolved in water were aseptically added to cultures of Escherichia coli and Bacillus subtilis grown on agar plates and incubated for 24 h at 37 °C. The initial inoculum for culturing both bacteria on Luria–Bertani (LB) agar medium was 10⁵ CFU mL⁻¹ prepared in LB broth at pH = 7.0. The antibacterial activity was determined by calculating the extended diameter of the zone of inhibition following the standardized guideline of the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). Inhibition zones with a diameter of 15–16 mm were considered as intermediately sensitive; inhibition zones with a diameter of ≥17 mm as sensitive; and inhibition zones with a diameter of <13 mm were considered as resistant.

2.11 Statistical analysis

We performed all biological experiments in triplicate and repeated them five times. We analysed differences among the data statistically employing Student's t-test and a value of p < 0.05 was considered as statistically significant.

3. Results and discussion

3.1 Description of SHGel-capped Ag NPs

The microstructure of SHGel-capped Ag NPs was investigated by TEM analysis (Fig. 1). EDX spectra were collected from different regions using high-angle annular dark-field scanning/transmission electron microscopy (HAADF-STEM) mode to obtain elemental information (Fig. 1). The spectra reveal the presence of C, O, and Ag elements. The Cu signal is obtained from the grid, and the C signal is due to the carbon-coated grid and the SHGel network that stabilized the Ag NPs, which was confirmed by EFTEM imaging (Fig. 1). The STEM-HAADF images (Fig. 1e) confirm that the SHGel network acted as an effecting capping agent for the Ag NPs. The TEM analysis given in Fig. 1f and g shows that the average diameter of the silver particles generated in situ is ∼20 nm. The TEM image shown in Fig. 1h illustrates the crystalline nature of SHGel-capped Ag NPs. Dynamic light scattering (DLS) studies and the zeta potential distribution of SHGel-capped Ag NPs in water are given as Fig. S1 in the ESI.†

Fig. 1 SHGel-capped Ag NPs. (a) and (b) TEM images showing the microstructure of Ag nanoparticles capped with SHGel. (c) STEM-HAADF image of SHGel-capped Ag NPs. (d) EDX spectrum from the region marked as area 1 in (c). (e) STEM-HAADF-EDX images taken from the area marked as area 2 in (c), indicating the locations of different atoms across the structure. (f) Size distribution of SHGel-capped Ag NPs. (g) Graphical representation of the size distribution pattern based on (f). (h) Crystalline nature of SHGel-capped Ag NPs.

The luminescent SHGel framework²⁴ has been treated as a stabilizing matrix for the sunlight-directed synthesis of silver nanoparticles (Fig. 1). Owing to the luminescent stabilizing agent (i.e., SHGel), SHGel-capped Ag NPs are also fluorescent with a fluorescence emission maximum at 365 nm (λ_ex = 300 nm) at 298 K and atmospheric pressure, which is evident from fluorescence spectra and microscopy studies (see ESI, Fig. S2†).

Ag NPs were photochemically generated from silver nitrate under direct exposure to sunlight without any externally added reducing chemicals and the synthesized Ag NPs became stabilized by the luminous nontoxic SHGel network.²⁴ Therefore, luminous SHGel-capped Ag NPs might also be biologically relevant. The luminescent SHGel network in its swollen state offers an enormous exposed space that might be available for the nucleation, specific growth and necessary stabilization of Ag NPs.²⁷ According to the principles of green chemistry,²⁶ this work possibly provides a green synthetic approach to the sunlight-mediated generation of nontoxic Ag NPs.

3.2 Drug transport and cellular incorporation of SHGel-capped Ag NPs

SHGel-capped Ag NPs might be very effective for drug transport into cells. With this aim in mind, we tried to investigate the stability of nanoparticle–drug composites consisting of drugs and Ag NPs, and determine the cellular incorporation capacity of SHGel-capped Ag NPs using macrophages from Wistar rats (Rattus norvegicus).

Several selective drugs such as streptomycin (a broad-spectrum antibacterial antibiotic), albendazole (a broad-spectrum anthelmintic), ivermectin (an insecticide and antifilarial) and diethylcarbamazine (an antifilarial) were employed to produce four nanoparticle–drug composites of the respective drugs and SHGel-capped Ag NPs. Confirmation that the nanoparticle–drug composites were formed is the key aspect for the drug transport capacity of SHGel-capped Ag NPs. Infrared spectroscopic analysis experimentally concluded that the Ag NPs chemically interacted with these drugs (see ESI, Fig. S3–S6†), which also supported the formation of nanoparticle–drug composites. The nanoparticle–drug composites also suggest the possibility of drug transport using SHGel-capped Ag NPs.

The cellular incorporation capacity of SHGel-capped Ag NPs was also investigated using macrophages from Wistar rats (Rattus norvegicus) following microstructural evidence. The cellular incorporation of hydrogel-capped Ag-NPs has been demonstrated via TEM analysis (Fig. 2). The results of TEM microstructural and the corresponding EDX spectral analysis (Fig. 2d) indicate that SHGel-capped Ag NPs can easily penetrate cell membranes and accumulate within cells. Therefore, the existence of nanoparticle–drug composites and the entry into cells of the Ag NPs support the proposed idea of drug transport using SHGel-capped Ag NPs.

Fig. 2 (a) and (b) Macrophage treated with SHGel-capped Ag NPs with a concentration of 2.5 μg mL⁻¹. (c) and (d) STEM-HAADF image and corresponding EDX spectrum from the region marked as 1 in (c).

3.3 Toxicity analysis of SHGel-capped Ag NPs

Cytotoxicity studies of SHGel-capped Ag NPs were carried out in vitro (by an MTT assay with rat peritoneal macrophages) and in vivo (using a Wistar rat model) (Fig. 3 and see ESI, Fig. S7 and Table S1†). In vitro and in vivo studies confirmed that SHGel-capped Ag NPs are nontoxic and biocompatible.

Fig. 3 Determination of mortality of rat peritoneal macrophages in the presence of different doses of SHGel-capped Ag NPs by MTT assay.

Because both Ag NPs are bioactive, it was necessary to determine their noxious effects on mammalian cells, i.e., macrophages, as well as whole organisms, i.e., rats. In both cases, we observed no detectable toxicity, which suggests that the activity of Ag NPs is specifically directed towards microorganisms (E. coli, B. subtilis and P. guilliermondii) and parasites (S. cervi). Previously, our study³¹ demonstrated that bioactive Ag NPs exerted no detectable cytotoxic effects on macrophages isolated from rats in vitro, in which the cellular morphologies were not altered either. Our study also corroborated this previous study³¹ and extended to an in vivo examination. The proportions of WBC, neutrophils, lymphocytes, basophils, eosinophils, monocytes and haemoglobin, which are haematological parameters, are typical indicators of toxicity at the haematological level, whereas bilirubin, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase are markers of liver function.³² Any deviation from their normal level upon the administration of any analyte indicates toxicity of the test analyte.³² However, neither of our synthesized Ag NPs altered the normal levels of the aforesaid parameters, which indicated the nontoxic nature of the particles in vivo and was also supported by another previous report.³³

3.4 CD spectroscopic analysis of SHGel-capped Ag NPs with CT-DNA

Owing to the addition of SHGel-capped Ag NPs to an aqueous solution of CT-DNA, the intensity of the CD spectroscopic signal of CT-DNA was altered (Fig. 4). The positive band at 277 nm indicates base stacking, whereas the negative band at 243 nm signifies helicity, which is the characteristic marker of right-handed B-DNA.^34–36 These sensitive bands are the principal markers for studying the interaction of ligands with DNA.^37,38 Intercalation of a species with DNA can alter the topology of DNA, but minor groove binders do not affect the CD spectrum of DNA significantly.^37,38 There were no observable changes in the CD spectrum, which confirms the absence of intercalation, but the presence of groove binding with DNA strands leading towards a reduction in helicity (Fig. 4). CD spectral data (Fig. 4) clearly show that there are non-covalent-type (i.e., minor groove) binding interactions between SHGel-capped Ag NPs and CT-DNA in an aqueous medium.^34–38 Minor groove binding interactions were revealed by the absence of characteristic changes, with the exception of intensity, in the CD spectrum, which demonstrates decoiling of the DNA double helix after exposure to SHGel-capped Ag NPs under ambient conditions.

Fig. 4 CD spectra of CT-DNA with and without SHGel-capped Ag NPs.

3.5 Biofunctionality of SHGel-capped and DNA hydrogel-capped Ag NPs

The biocompatible features of both SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs²⁴ motivated us to determine the biofunctionality of these silver nanomaterials against pathogenic microorganisms and parasites. Furthermore, the absence of bioactivity of the nanoparticle-stabilizing scaffolds, i.e., SHGel and DNA hydrogel,²⁴ is an additional advantage for exclusively obtaining the distinctive biofunctionalities of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs.²⁴

3.5.1 Antifilarial activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs. To study the antifilarial activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs, Setaria cervi, which is a model filarial parasite (including both macro- and microfilariae), was employed. These two different nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs) markedly affected the viability of parasites in a fixed population in a dose-dependent manner (Fig. 5 and 6).

Fig. 5 Antifilarial activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag-NPs against Setaria cervi microfilariae: (i) control, (ii) SHGel-capped Ag NPs with a concentration of 5 μg mL⁻¹, (iii) DNA hydrogel-capped Ag NPs with a concentration of 2.5 μg mL⁻¹.

Fig. 6 Determination of parasite mortality by an MTT assay. Adults and microfilariae of S. cervi were exposed to increasing concentrations of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs for 24 h. Data are shown in triplicate (mean ± SD).

Although both silver nanoparticles are efficient antifilarial agents, the previously reported DNA hydrogel-capped Ag NPs²⁴ are more effective and this is also shown by the LC₅₀ values obtained for SHGel-capped Ag NPs and DNA hydrogel-capped Ag-NPs (i.e., 2.42 and 3.7 μg mL⁻¹ for macro- and microfilariae, respectively, for SHGel-capped Ag-NPs and 1.97 and 2.56 μg mL⁻¹ for adults and mf, respectively, for DNA hydrogel-capped Ag NPs) (Fig. 7). The death of the parasites might be achieved via apoptosis, as found by acridine orange/ethidium bromide staining in fluorescence microscopic imaging (Fig. 5).

Fig. 7 LC₅₀ values of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs. LC₅₀ values for adult (a) and microfilarial (b) parasites exposed to SHGel-capped Ag NPs were 2.42 and 3.7 μg mL⁻¹, respectively. LC₅₀ values for adult (c) and microfilarial (d) parasites exposed to DNA hydrogel-capped Ag NPs were 1.97 and 2.56 μg mL⁻¹, respectively. LC₅₀ values were calculated from the plots shown above.

3.5.2 Fungicidal activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs. These two synthesized nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs) were also effective against the pathogenic fungus Pichia guilliermondii, which was isolated from a subject with microfilaraemia (see ESI, Fig. S8†). The fungicidal effect of these two silver nanoparticles was evident with MIC values of 2.5 and 1.25 μg mL⁻¹ for SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs, respectively (Fig. S8†), which confirmed the superior efficiency of DNA hydrogel-capped Ag NPs over SHGel-capped Ag NPs as a fungicide (Fig. 8).

Fig. 8 Zones of inhibition found in bacterial and fungal culture plates generated by the action of (a) SHGel-capped Ag NPs and (b) DNA hydrogel-capped Ag NPs.

3.5.3 Bactericidal activity of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs. The antibacterial activities of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs are remarkable. Both nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs) also exert a potent bactericidal effect against Gram +ve (Bacillus subtilis) and Gram −ve (Escherichia coli DH5α) bacteria (see ESI, Fig. S9†). Based on the zones of inhibition (Fig. S9†) after treatment with the two types of Ag NPs at different dosages, it was observed that both SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs exhibit potential antibacterial features in a dose-dependent manner.

Here, the particle behaviour of the synthesized Ag NPs was tested on biological systems. Interaction between Ag NPs and CT-DNA was evident in CD spectroscopic analysis. This interaction could be the reason behind the excellent bioactivity of these Ag NPs, because DNA is the genetic material of the bacteria and fungus that we tested. Such an assumption or rather interpretation that a silver nanoparticle-induced alteration in the structure of DNA contributes to the bioactivity of Ag NPs has been supported by a previous study.³¹ Moreover, the Ag NPs investigated in this study act at a relatively lower dose compared with previous reports on nontoxic Ag NPs.³¹

3.6 Bacterium (Bacillus subtilis)-directed comparative nano-patterning feature

Most interestingly, during treatment with SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs against Gram +ve bacteria (Bacillus subtilis), two different thin-film-like patterns were generated and assembled on the paper discs containing primary nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs), as shown in Fig. S9 in ESI.† TEM microstructural analyses, along with EDS studies, confirmed that the two abovementioned different thin-film-like nanosilver patterns originated during the action of SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs against the Gram +ve bacteria (B. subtilis) (Fig. 9 and 10). Cotyledon-shaped Ag nanoparticles were generated by the action of SHGel-capped Ag NPs against Gram +ve bacteria (B. subtilis), as found by TEM analysis (Fig. 9). Flower bud-shaped Ag nanoparticles were also produced by the action of DNA hydrogel-capped Ag NPs on Gram +ve bacteria (B. subtilis), as observed by TEM analysis (Fig. 10). Therefore, comparative nano-patterning features directed by the bacterium Bacillus subtilis in SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs have been established.

Fig. 9 Silver nanoparticles originating from the action of SHGel-capped Ag NPs against B. subtilis. The silver nanoparticles are observed from the patterns shown in Fig. S9 in ESI† for 2.5 μg mL⁻¹ SHGel-capped Ag NPs used for treatment against B. subtilis. (a) TEM image showing the microstructure of cotyledon-shaped Ag nanoparticles originating from the action of SHGel-capped Ag NPs against B. subtilis. (b) STEM-HAADF image of cotyledon-shaped Ag nanoparticles. (c) EDX spectrum from the region marked as area 1 in (b) confirming the generation of cotyledon-shaped Ag NPs. (d) STEM-HAADF-EDX images taken from the area marked as area 2 indicating the locations of different atoms across the structure. Here, the Cu signal in the EDX spectrum is due to the grid. The O signal in the EDX spectrum with the STEM-HAADF-EDX image (O-K) is due to the hydrogel (i.e., SHGel), which acted as a capping agent for Ag NPs. The presence of an S signal in the EDX spectrum, which is evident for the pattern of cotyledon-shaped silver nanoparticles, originated from the action of SHGel-capped Ag NPs against B. subtilis.

Fig. 10 Silver nanoparticles originating from the action of DNA hydrogel-capped Ag NPs against B. subtilis. The silver nanoparticles are observed from the patterns shown in Fig. S9 in ESI† for 1.25 μg mL⁻¹ DNA hydrogel-capped Ag NPs used for treatment against B. subtilis. (a) STEM-HAADF image showing the microstructure of flower bud-shaped Ag nanoparticles originating from the action of DNA hydrogel-capped Ag NPs against B. subtilis. (b) STEM-HAADF image of flower bud-shaped Ag nanoparticles. EDX spectra from the region marked as area 1 in (a) and the area denoted as 2 in (b), which confirm the formation of flower bud-shaped Ag NPs. Here, the Cu signal in the EDX spectrum is due to the grid. The presence of an S signal in the EDX spectrum, which is evident for the pattern of flower bud-shaped silver nanoparticles, originated from the action of DNA hydrogel-capped Ag NPs against B. subtilis.

4. Conclusion

In brief, we have established a green synthetic method for the generation of silver nanoparticles. Ag NPs have been photochemically synthesized by direct exposure to sunlight without any externally added reducing chemicals. A nontoxic luminous supramolecular hydrogel network²⁴ was employed as a stabilizing scaffold for the synthesized silver nanoparticles. Owing to the luminous capping agent (i.e., SHGel²⁴), the Ag NPs should be luminescent and this hypothesis has been confirmed via the fluorescence spectra and microscopic analyses of SHGel-capped Ag NPs. In vivo and in vitro toxicity tests show the biologically relevant and nontoxic nature of SHGel-capped Ag NPs. Nontoxic SHGel-capped Ag NPs might be effective for drug delivery into cells and this theory of drug delivery using SHGel-capped Ag NPs was confirmed by testing the cellular incorporation ability of SHGel-capped Ag NPs and investigating the stability of drug–nanoparticle composites. TEM microstructural and EDX spectral analyses show the cellular incorporation capacity of SHGel-capped Ag NPs. In addition, the interactions between different selective drugs, including streptomycin (a broad-spectrum antibacterial antibiotic), ivermectin (an insecticide and antifilarial), albendazole (a broad-spectrum anthelmintic) and diethylcarbamazine (an antifilarial), and SHGel-capped Ag NPs have been experimentally proved via infrared spectral analysis. The results of infrared spectroscopy confirm the formation and stability of nanoparticle–drug composites. Therefore, the potential drug transport capacity of SHGel-capped Ag NPs has been established.

Moreover, the non-bioactivity of SHGel²⁴ and the nontoxic behaviour of SHGel-capped Ag NPs encouraged us to test the bioactivity of SHGel-capped Ag NPs. In addition to SHGel-capped Ag NPs, the bioactivity of previously reported DNA hydrogel-capped Ag NPs²⁴ has also been tested by a similar approach. Both nanoparticles (i.e., SHGel-capped Ag NPs and DNA hydrogel-capped Ag NPs) are potentially bioactive in terms of antifilarial, antifungal, and antibacterial activities.

Most remarkably, we have investigated the functionality of Gram +ve bacteria (i.e., Bacillus subtilis) for the formation of two different nano-scale silver films, which were generated during the action of SHGel- and DNA hydrogel-capped silver nanoparticles against the bacterium Bacillus subtilis. TEM microstructural analysis shows that cotyledon- and flower bud-shaped Ag NPs were generated separately by the action of SHGel-capped Ag NPs and DNA hydrogel-capped Ag-NPs,²⁴ respectively, against the bacterium Bacillus subtilis.

Acknowledgements

B. D. is thankful to DST (New Delhi, India) for a research project (Project No. SR/FT/CS-77/2011) for financial support. S. P. S. B. is also thankful to CSIR, Govt. of India for a research grant (37 (1516/11/EMR-II)) for financial support.

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Footnote

† Electronic supplementary information (ESI) available: Dynamic light scattering study and zeta potential distribution of SHGel-capped Ag NPs in water, fluorescence spectra and fluorescence micrograph of SHGel-capped Ag NPs, infrared spectral data for evaluating the formation of drug–SHGel-capped Ag NPs composites, hematological and serological parameters for toxicity evaluation. See DOI: 10.1039/c5ra27886d

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