Biocompatible nickel-prussian blue@silver nanocomposites show potent antibacterial activities

Aim: Silver nanoparticles have long been used as potent antibacterial agents. However, toxicity concerns of silver nanoparticles have limited their successful clinical applications. Hence, development of silver-based novel biocompatible nanomaterials for antibacterial applications is a challenging task. Materials & methods: Accordingly, in this work, we synthesized a biocompatible silver-based nanocomposite for antibacterial applications. The nanocompostie was characterized by several analytical techniques. The nanocomposite was further tested for its cytotoxicity in cells, chicken embryo and bacteria. Results & Conclusion: Herein, we report a simple and cost-effective method for the synthesis of nickel-prussian blue@silver nanocomposites. The nanocomposite is highly stable and shows biocompatibility observed by in vitro assay and by ex vivo chicken embryonic angiogenesis assay. The nanocomposite exhibits profound antibacterial activity toward Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtillus) bacteria. The results altogether suggest the future potential applications of nickel-prussian blue@silver nanocomposite as an antibacterial agent.


Cell lines
Human lung cancer cell lines (A549) and mouse melanoma cell lines (B16F10) were purchased from American Type Culture Collection (ATCC). Human endothelial somatic hybrid cell lines (EA.hy926) were a kind gift from S Oglesbee, Tissue Culture Facility, University of North Carolina, Lineberger Comprehensive Cancer Center, NC, USA, and S Chatterjee, Anna University -K B Chandrasekhar, Chennai, India.

Synthesis of AgNPs
AgNPs were synthesized by borohydride reduction methods according to the published literature [12]. Briefly, 25 ml of AgNPs was prepared by the reduction of 1 ml of AgNO 3 (10 -2 M) in 10 ml of water using 14 ml of NaBH 4 (0.05 mg/ml) for 1 h under continuous stirring condition.

Stability studies of NiPB@AgNC in various physiological buffers & salines
The stability of any nanomaterials is very important before its use in healthcare. In vitro stability of NiPB@AgNC was investigated in 10% NaCl solution and different PBS with different pH (6, 7.4 and 8) for 2 weeks. Briefly, 100 μl of NiPB@AgNC pellet was incubated with 900 μl of respective buffers and kept for 14 days. The absorbance and λ max of the corresponding mixtures was recorded in UV-Visible spectroscopy in a time-dependent manner. Finally, we plotted the λ max of NiPB@AgNC (wavelength corresponds to maximum absorbance) incubated in various buffers in different time points and compared the stability of the nanocomposites. Additionally, we measured the hydrodynamic diameter of NiPB@AgNC incubated in various buffers using dynamic light scattering (DLS: Zetasizer Ver. 6.20; Malvern-MAL100442) in different time points to check the stability of the nanocomposites.

Cell culture experiments
All cancer cell lines [A549 and B16F10] and normal cell line (EA.hy926) were cultured in DMEM media supplemented with 10% fetal bovine serum, 0.001% antibiotics (penicillin-streptomycin-kanamycin) and 5% L-glutamine in a humidified 5% CO 2 incubator at 37 • C for all in vitro biological experiments. The cells were incubated with different concentrations of NiPB@AgNC after 30 min of exposure under UV light.
Cell viability test using MTT reagent Initially, 10 × 10 3 cells (EA.hy926, A549 and B16F10) were seeded in a 96-well plate and kept for 24 h in cell culture conditions (humidified 5% CO 2 incubator at 37 • C). Cell viability of all cancer (A549 and B16F10) and normal cells (EA.hy926) were carried out using MTT reagents after incubation of cells with different doses of NiPB@AgNC (1, 2.5, 5 and 7.5 μl/ml correspond to [Ag] = 14, 35, 70 and 105 μM, respectively) for 24 h according to our published protocol. After 24-h incubation, all treated cells were washed with PBS to remove surface-bound nanoparticles, and subsequently 100 μl of MTT solution (0.5 mg/ml in DMEM media) was added into each well for further 4 h. After 4 h of incubation, the MTT solution was replaced by freshly prepared 100 μl of DMSO:MeOH (1:1, v/v) in each well and absorbance were measured at λ max = 575 nm. Finally, the results were expressed as normalized percent cell viability = ([A570 {treated cells} -background]/[A570 {untreated cells}background]) × 100.

Confocal microscopic analysis for fluorescent cytoskeleton & nucleus observation
To assess the morphology of the cells by polymerized actin, following fixation cells were analyzed by confocal microscopy. First, cells were grown on a 60-mm dishes on embedded cover slips at a density of approximately 50,000 NIH-3T3 cells/well (obtained from NCCS, Pune, India). After  Chick embryonic angiogenesis assay The chicken embryonic angiogenesis (CEA) assay, a standard ex vivo angiogenesis assay, has been widely used to study angiogenesis and tumor invasion of different cancers [24,25]. The fertilized chicken eggs were purchased from government poultry (Directorate of Poultry Research, Hyderabad, Telangana) and incubated in an egg incubator (Southern) at 37 • C and 60-70% relative humidity. On the fourth day of incubation, a small gap was created on the shell using forceps carefully, sterilized filter-paper discs were soaked in the NiPB@AgNC pellet ([Ag] = 3.5, 7, 14 μM). The discs were employed around the blood vessels after exposing the gap area that was covered with parafilm later. The treated and untreated eggs were incubated for next 4 h in the incubator. The images were captured at 0 and 4 h post-treatments using an LEICA camera (LEICA MC120-HD) attached to a LEICA stereomicroscope (LEICA S8AP0) at 10 megapixel magnifications.

Antibacterial activity of NiPB@AgNC
Gram-negative E. coli and Gram-positive B. subtillus growth was maintained at 37 • C in shaker in Luria bertani broth media. The inhibition growth of E. coli and B. subtillus treated with different doses of NiPB@AgNC (1, 2.5 and 5 μl/ml corresponding to [Ag] = 14, 35 and 70 μM; respectively) was monitored by multimode spectrophotometer (Varioskan) at 600 nm up to 6 h. Streptomycin (100 μg/ml) was used as positive control antibiotics. Agar disc diffusion technique was used to evaluate antibacterial activity of E. coli and B. subtillus against nanocomposite and standard antibiotics like streptomycin (100 μg/ml) and penicillin (100 μg/ml) by measuring the inhibitory zone.

Release of silver ions from NiPB@AgNC in PBS
Release of silver ions from nanocomposite was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. Initially, 500 μl of the ash yellow pellet of NiPB@AgNC was obtained after centrifugation of 50 ml of reaction mixture at 17,700 r.p.m. 15 • C for 40 min. 100 μl of the pellet of the nanocomposite was incubated with 900 μl of PBS (pH 7.4) and kept for different time points (0-48 h). After each time point, the mixture was mixed with 20 ml of Milli-Q water and the solution was ultracentrifuged at 24,000 r.p.m. for 1 h at 20 • C. The supernatant of each solution was collected and further submitted to ICP-OES analysis for the measurement of released silver content.

Characterization techniques
The as-synthesized nanocomposite was thoroughly characterized by several physicochemical techniques. Initially, 50 ml of NiPB@AgNC (obtained after second step of reaction) was centrifuged for 40 min at 17,700 r.p.m. at 15 • C using ultracentrifuge (Thermo Fisher Scientific, Sorvall-WX ultra 100). The intense loose ash yellow-colored pellet obtained after centrifugation was used for further characterizations and cell viability studies and antibacterial studies. The absorbance, crystallinity and purity of NiPB@AgNC were monitored by UV-Visible spectroscopy (JASCO dual-beam spectrophotometer [Model V-570]) and x-ray diffraction (XRD) analysis using Bruker AXS D8 Advance Powder x-ray diffractometer (CuKαλ = 1.5406Å radiation), respectively. The size, morphology and surface charge (zeta potential) were determined by transmission electron microscopy (TEM: FEI Tecnai F12, Philips Electron Optics, Eindhoven, Holland) and DLS, respectively. ICP-OES [(iCAP-6500 DUO), Thermo Fisher Scientific, Cambridge, UK] was used for the determination of silver, nickel and iron in the loose pellet.

Results & discussion
The synthesis of NiPB@AgNCs was carried out by simple addition of freshly prepared AgNPs with the mixture of equal volume of K 3 [Fe(CN) 6  UV-visible spectroscopy UV-visible spectroscopy was used to monitor the formation of NiPB@AgNC. Figure 2A shows the UV-visible spectrum of K 3 Fe(CN) 6 , naked AgNPs and NiPB@AgNCs obtained at first and second step of reaction (NiPB@AgNC-1 and NiPB@AgNC-2). The absorbance of NiPB@AgNC-1 and NiPB@AgNC-2 was monitored at λ max approximately 410-415 nm indicating the possibility of formation of NiPB@AgNCs, which differs from the UV spectrum of K 3 Fe(CN) 6 and AgNPs ( Figure 2A). The absorbance intensity of NiPB@AgNC-2 is more compared with NiPB@AgNC-1, indicating the higher concentration of NiPBNPs after second step ( Figure 2A). Figure 2B shows the optical images of the color of the K 3 [Fe(CN) 6 )] (light yellow), AgNPs (intense ash), NiPB@AgNC-1 (ash yellow) and NiPB@AgNC-2 (intense yellow), respectively, which emulate the plausible stepwise formation of nanocomposites.

XRD spectroscopy
The crystal structure of NiPB@AgNC and NiPBNPs was identified by XRD analysis ( Figure 2C & D). The diffraction peaks observed for both NiPB@AgNC and NiPBNPs at 200, 220, 400, 420, 440, 600 and 620 indicate face-centered cubic (Fm-3m) crystalline lattice structure [26,27] (Figure 2C & D). This result corroborates with the XRD pattern of well-established prussian blue-based nanoparticles and nanocomposites [26,27]. However, we did not find any separate diffraction peaks for AgNPs in the XRD spectra of NiPB@AgNC. This is probably due to the double-layer thick coating of NiPBNPs on AgNPs that mask the indicative peaks of AgNPs in the XRD spectra of NiPB@AgNC. This also might be due to the poor crystallinity of AgNPs [28]. XRD results further reveal that there is no change of crystal structure of basic moiety (NiPBNPs) after formation of the nanocomposites.

Size, shape & morphology analysis
In order to find out the size and morphology of nanoparticles and nanocomposite (NiPB@AgNC), TEM analysis was carried out. Figure 2E shows the TEM images of NiPBNPs. TEM images of NiPBNPs show the cubic shape with size range approximately 50-75 nm, whereas AgNPs show mainly spherical shaped with size range of 20-40 nm (data not shown) [12]. On the other hand, the TEM images of NiPB@AgNC show nice spherical bimetallic nanocomposite structures with the size range of approximately 100-120 nm, which are ideal for the biomedical applications ( Figure 2F). The DLS method was employed to calculate the hydrodynamic radii of nanocomposite.
Result shows that the hydrodynamic diameter of NiPB@AgNC is approximately 162 ± 5.2 nm ( Figure 2G). Again, the surface charge or zeta potential (ξ) is a crucial parameter for the dispersion or colloidal stability of nanoparticles. The size of the nanocomposite obtained from DLS is slightly higher compared with size obtained from TEM. DLS studies give an idea about the hydrodynamic diameter of any nanoparticles, which includes the association of water other molecules, whereas TEM shows the exact size of nanoparticles excluding the surface-attached molecules. The ξ value of NiPB@AgNC is highly negative (-27.1 ± 4.34 mV), indicating exceptional stability due to high-repulsive surface negative charge ( Figure 2H).

Fourier transformed infrared spectroscopy
The peak at 2098 cm -1 in Fourier transformed infrared (FTIR) spectrum of NiPB@AgNC is indicating the presence of C≡N stretching frequency ( Figure 3A) [29]. The lower stretching frequency of C≡N might occur due to the interaction of nickel with cyanide (C≡N) in the lattice state of NiPBNPs [3,29]. The cyanobridged complexes can easily be identified as they exhibit sharp peak at ν(CN), at 2000-2200 cm -1 [30]. The two peaks at 2098 and 2165 cm -1 arose owing to the transformation of the microstructure Fe 2+ -CN-Ni 3+ to the structure Fe 3+ -CN-Ni 2+ . The peaks at 2098 and 2165 cm -1 correspond to the ν(Fe 2+ -CN-Ni 3+ ) and ν(Fe 3+ -CN-Ni 2+ ), respectively. More importantly, the major part of the complex contains trivalent ion rather than the bivalent ion. The FTIR peaks at 3412 and 1611 cm -1 designated to the O-H stretching and O-H bending vibrations of the crystal water in the framework of the nanocomposite, respectively [31,32]. The stretching frequency of the -COO bond comes at 1416 cm -1 , which is less intense as nickel acetate is used as one of the precursor. Meanwhile, in the low-frequency region, the peak at 595 cm -1 corresponded to the Ni-O vibration in the NiO 6 coordinate sphere [31,33].

Stability studies
The stability of nanoparticles/nanocomposites is vital before its use in biomedical applications. Therefore, in vitro stability of nanocomposite was investigated in 10% NaCl solution and PBS with different pH (6, 7 and 8) for 2 weeks. Figure 3B shows that the maximum wavelength (λ max ) of NiPB@AgNC in different buffers remains almost unchanged (<5 nm) after 2 weeks of incubation with respect to 24-h sample, which reflects the high colloidal stability. Here, it is important to mention that salt stability is an important parameter to determine the stability and to test the aggregation properties of nanoparticles. Several groups including ours have tested the in vitro stability of AgNPs/nanocomposites in salt or saline solutions [12]. However, it is vital to mention that bare AgNPs may show formation of AgCl in saline solutions with time and subsequent decrease in the absorbance (sign of aggregation) of the nanoparticles that indicate instability. In our case, we do not found any decrease in the absorbance of the nanocomposites incubated with saline solutions till 14 days. This indicates the stability of NiPB@AgNC in salt solutions. This is probably due to the external double-layer thick coating of NiPBs on AgNPs, which prevents the quick dissolution of AgNPs and subsequent formation of AgCl in salt solutions. Moreover, we measured the hydrodynamic size of NiPB@AgNC in these buffers till day 14 (Table 1). From the Table 1, it is clear that no significant change in the hydrodynamic size of NiPB@AgNC incubated in different buffers has occurred till day 14. This directly supports the in vitro stability of NiPB@AgNC in various buffers (with pH 6-8) and saline solutions.

In vitro cytotoxicity & morphological analysis
For successful application of NiPB@AgNC in biology and medicine, it is very important to check their potential cytotoxicity. In vitro cell viability assay using MTT reagents is a basic assay to evaluate the cytotoxicity of any nanomaterials or drugs. In order to check the cytotoxicity of nanocomposites, we carried out in vitro cell viability assay using MTT reagents in various cancer (A549 and B16F10) and normal cell lines (EA.hy926) for 24 h   in a dose-dependent manner ( Figure 4A-C). Cell viability results show that NiPB@AgNC is biocompatible in nature up to high doses of silver ([Ag] = 105 μM) in both normal as well as cancer cells ( Figure 4A-C). The biocompatibility of the synthesized nanocomposite could make it useful for further biomedical applications. Our previous report demonstrates that AgNPs show cytotoxicity at 30 μM dose in various normal cell lines [12]. In this case, the nanocomposite is biocompatible even at higher concentration of silver ([Ag] = 105 μM), indicating the biocompatibility of this material up to high doses.
future science group www.futuremedicine.com To provide further evidence of biocompatibility and visualization of cell morphology upon treatment with nanocomposites, confocal microscopy was used. The cell morphology and observation of fluorescent cytoskeleton and nucleus also give the idea of the biocompatibility of the nanoparticles or nanocomposite-treated cells. Staining of the actin filaments and nucleus give the overall structure of the cells. Results reveal that there is no change in the overall morphology of the NIH-3T3 cells even in the treatment of high doses of NiPB@AgNC (7.5 μl/ml; correspond to [Ag] = 105 μM for 24 h) compared with the untreated cells ( Figure 5A-H). The pictures clearly show that there is no change in the actin cytoskeleton structure or presence of nuclear fragmentation even after high doses of treatment ( Figure 5A-H). These results indicate the biocompatibility of nanocomposites even at high doses [34,35].

CEA assay: ex vivo toxicity
The CEA assay, a typical ex vivo angiogenesis assay, has been commonly used to study angiogenesis and ex vivo toxicity [24,25]. In order to investigate the ex vivo biocompatibility of the nanocomposite we performed the CEA assay. Figure 6 indicates the CEA assay where egg yolk was untreated control (control: Figure 6), or treated with NiPB@AgNC at different concentrations (3.5, 7 and 14 μM). The incubation was done for 4 h. From the CEA data, no remarkable change in the blood vessels is found in different doses. The CEA results further support the ex vivo biocompatible nature of the nanocomposite, which could be useful for the safe use of these nanomaterials in healthcare and medicine after proper preclinical study. Hence, both in vitro and ex vivo results support the nontoxic nature of NiPB@AgNC.

Antibacterial activity Growth inhibition study
Time-dependent growth of untreated bacteria (E. coli and B. Subtillus) and bacteria treated with different doses of NiPB@AgNC in Luria bertani broth was studied by measuring the O.D. of bacteria. Figure 7A & B shows the graphical representation of inhibition kinetics for E. coli and B. subtillus treated with different concentrations of nanocomposite. The inhibition of growth is observed with NiPB@AgNC in a dose-dependent manner compared with untreated bacteria. Around 100% inhibition is observed at 70 μM of Ag (5 μl/ml of NiPB@AgNC) at 6 h, which is similar with the effects of positive-control drug streptomycin (100 μg/ml), indicating high therapeutic efficacy.

Zone of inhibition study
Agar diffusion disc method was employed in order to investigate the antibacterial activity of different concentration of NiPB@AgNC toward E. coli and B. subtillus. The antibacterial activity of NiPB@AgNC was compared with standard antibiotics like streptomycin (100μg/ml) and penicillin (100 μg/ml) by measuring the zone of inhibition (Table 2, Figure 8A & B). The results are tabulated in Table 2  NiPB@AgNC: Nickel-prussian blue@silver nanocomposite; ZOI: Zone of inhibition. (∼14 mm; Figure 8A & B). Again, NiPB@AgNC shows almost comparable inhibition zone (∼16 mm) with standard antibiotics streptomycin (∼18 mm) in Gram-positive bacteria, B. subtillus ( Figure 8A & B). Our earlier published reports show that AgNPs exhibit around 10 mm of inhibition zone toward E. coli at 30 μM of silver concentration [12]. Compared with earlier published reports, it is obvious that the nanocomposites exhibit twice the antibacterial activity (∼20 mm zone of inhibition at [Ag] = 14 μM), at half of the silver concentration compared with AgNPs. The nanocomposites in the present work thus show far superior antibacterial activity compared with AgNPs, establishing the elevated therapeutic efficacy of these materials.
future science group www.futuremedicine.com It is well established that AgNPs show excellent antibacterial activity due to mainly bacterial membrane damage or production of reactive oxygen species by Ag + ions [12]. To get a possible idea regarding the mechanism of antibacterial activity of the nanocomposites, we checked the release of silver ions in a time-dependent manner. Results show that a time-dependent release of Ag + ions from NiPB@AgNC, which get saturated after 60 min and continue to sustain release till 48 h ( Figure 8C). This sustained release of Ag + ions could be the plausible reason for the antibacterial activity of the nanocomposites in Gram-negative and Gram-positive bacteria. Recently, Lu et al. exhibited that AgNPs convert to Ag + and superoxide anions in presence of dissolved oxygen in a rapid manner, according to the following reaction [36,37]: AgNPs + O 2 (dissolved oxygen) → Ag + (silver ion) + O 2 .-(superoxide radical) However, the release of Ag + ions from AgNPs is rapid and hence nonspecific in nature. Hence, rapid release of Ag + may cause toxicity due to lack of targeting ability, nonspecific deposition of silver and lack of stability. However, the release of Ag + from NiPB@AgNC is much slower than AgNPs, due to double-layer coating of NiPBNPs on AgNPs. Consequently, this prevents rapid and nonspecific leakage of Ag + ions. Hence, this sustained and slow release of Ag + ions from NiPB@AgNC ensures the enhanced therapeutic efficacy (antibacterial activity) and less cytotoxicity (in normal cells). Recently, different groups including ours have demonstrated development of multifunctional nanoparticles/nanocomposites for various theranostics applications [38][39][40]. Together, these above results strongly support the successful fabrication of a robust nanocomposite that can be used for bacterial theranostics applications.

Conclusion
In this article, we report the synthesis of NiPB@AgNCs. The synthesis method is simple, efficient, ecofriendly and economically cheap. Physicochemical characterizations of nanocomposite show high stability. The in vitro MTT assay in normal and cancer cells demonstrate the biocompatibility nature of nanocomposite. Ex vivo CEA assay further supports the biocompatibility of the nanomaterials. Finally, the nanocomposite exhibits excellent antibacterial activity against Gram-negative (E. coli) and Gram-positive (B. subtillus) bacteria. The results altogether demonstrate the future potential applications of this newly developed nanomedicine (NiPB@AgNC) as an effective antibacterial agent.

Future perspective
These exciting data on potent antibacterial activity of highly biocompatible silver-based nanocomposites are likely to pave the way for the development of potential safer nanomedicine that can be effectively used for clinical studies. Our synthesized nanomedicine (NiPB@AgNC) is safer at higher doses compared with several commercial silverbased nanomedicines. Moreover, NiPB@AgNC demonstrates significant high therapeutic activity. This could be a game changer in the near future considering low toxicity and improved therapeutic efficacy. However, detailed and rigorous studies still need to be explored, mainly regarding its mechanistic aspects and systematic in vivo toxicity before its clinical applications. Additionally, MRI studies also need to be performed to confirm the potential diagnostic ability of NiPB@AgNC for bacterial infections.

Summary points
r Nickel-prussian blue@silver nanocomposite (NiPB@AgNC) has been synthesized by a simple, efficient and cost-effective method. r NiPB@AgNC has been characterized by several analytical techniques (UV-visible spectroscopy, x-ray diffraction, dynamic light scattering and transmission electron microscopy, etc.). r The nanocomposite is highly stable in various physiological buffers and solutions for 2 weeks.
r NiPB@AgNC is biocompatible in nature, observed by in vitro (cellular morphological analysis, cytoskeleton analysis, nucleus analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and ex vivo chicken embryonic angiogenesis assay. r The nanocomposite exhibits potent therapeutic efficacy (antibacterial activity) against Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtillus) bacteria. r Mechanistic studies revealed that low cytotoxicity and improved antibacterial efficacy is due to the slow and sustained release of the silver ion (Ag + ).