e-ISSN: 2322-0139 p-ISSN: 2322-0120

Study of Toxicity and Antibacterial Effect of Silver Nanoparticles

Qureshi MZ, Hayee R* and Tahir S

Government College University, Lahore, Pakistan

*Corresponding Author:
Rabia Hayee
Pharmacist, Government College University
Lahore, Pakistan.
E-mail: rabiahayee1@gmail.com

Received date: August 09, 2017; Accepted date: August 22, 2017; Published date: August 29, 2017

Visit for more related articles at Research & Reviews: Journal of Pharmacology and Toxicological Studies

Keywords

Silver nanoparticles, XRD, SEM-EDX, FTIR, Bacillus subtilus, Albino mice, Microtomy

Introduction

Nanotechnology is rapidly growing area in the field of science by producing nano-size particles that can have novel physiochemical properties that may be different from the parent particles [1]. Among different nanoparticles, silver nanoparticles (AgNPs) attracted the major interest because of its unique physical, chemical and biological properties as compare to its macro scale particles [2]. The silver nanoparticles are widely used in biosensing, photonics, electronics, and antimicrobial applications [3-7]. Due to its strong antimicrobial activity, AgNPs are used for producing textiles, food storage containers, antiseptic sprays, catheters, and bandages [7]. AgNPs are found to possess the oligodynamic properties and can kill the antibiotic resistant microbes and also cause cytotoxic effect to mammalian cells [8-11].

The Ag+ ion concentration have been found to be responsible for toxicity of AgNPs [12,13], furthermore, different studies showed that AgNPs release the Ag+ ions in aerobic but not in anaerobic environment, and AgNPs’ antimicrobial properties also limited to aerobic environments [14]. The antimicrobial mechanism of AgNPs is as, Ag+ ions rupture cell walls of microbes, denaturing the cell proteins, blocking cell respiration, and ultimately inducing cell death [15-17], it has also been studied that the method of synthesis, size, shape and coating material on AgNPs may affect its toxicological properties [18,19].

Increasing trends of AgNPs indicate its adverse effects both in human and environment as well [20]. It has been reported that silver nanoparticles crossed cell membrane barriers to reach to different organs, started accumulate at site of absorption; caused toxicity and extensively cell death occur because of the nanoparticles’ small size and surface properties and exert the toxicity [21-27].

This present study had been done to study the antibacterial effect in gram positive and gram negative bacteria and toxicity of silver nanoparticles in male albino mice.

Materials and Methods

The chemicals used were of analytical grade brought from Acros Organics (Belgium), Merck (Germany) Fischer Scientific (UK) and Sigma Chemicals (USA) includes Silver Nitrate, Nutrient agar (NA), Nutrient Broth (NB), Luria Bertani (LB), Ethanol, 70% isopropanol, Sodium borohydride, Trisodium citrate, Soluble starch, Paraffin wax, Xylene, Stains (eosin, hemato-xylene) and Canada Balsam. The analytical equipment’s which were used in this study includes Analytical balance (EB-43-CH-Japan), Distillation plant, Hot plate, Oven (Memmert, Germany), Refrigerator (Laborata) UV-Visible Spectrophotometer (Shimadzu, UV-1700), Scanning Electron Microscope. Fourier Transform Infrared Spectrometer (MIDAC M 2000), Autoclave (ALP. CO. Ltd. 40 L, Japan), Incubator (Fisher Scientific, UK), Shaking incubator (Irmeco Gmbh, Germany). Laminar air flow (Technico Scientific, Lahore), Powder XRD and Compound Research Microscope. The microbes Bacilliis subtilis (gram positive) and Escherichia coli (gram negative), used for antibacterial activity were obtained from Bio-process laboratory, Department of Chemistry, GC University Lahore. The six male albino mice were obtained from Veterinary Research Institute (VRI), Lahore Cantonment. These were 4 weeks of age. They were kept for one week as their acclamation period.

Synthesis of silver nanoparticles

Starch encapsulation

Starch encapsulation is also known as polysaccharide method in which nanoparticles are synthesized in one-step. 1.0 g of soluble starch and 1 ml of 100 mM silver nitrate solution prepared in distilled water. The soluble starch was dissolved in 100 ml distilled water. It was then heated in an oven or a hot plate. 1 mL of a 100 mM aqueous solution of silver nitrate was mixed in the above solution and stirred well. This process was done with complete dissolution of starch into the solution. This mixture of two solutions was kept in an autoclave at 15 psi pressure, 121°C for 5 min. The resulting solution was clear yellow in color indicating The formation of silver nanoparticles was confirmed was solution was turned to light yellow color. The change in color indicated the formation of silver nanoparticles.

Collidal method

The chemical reduction of silver salts by sodium borohydride or sodium citrate is the most used method of preparation of silver colloids. The stable and reproducible colloids of silver nanoparticles can be synthesized if kept with great care Silver nitrate AgNO3 and trisodium citrate C6H5O7Na3 of analytical grade purity were used as starting materials without further purification. The silver colloid was prepared by using chemical reduction method (Sileikaite, 2006). In typical experiment 50 ml of 0.001 M AgNO3 was heated to boiling. To this solution 5 ml of 1% trisodium citrate was added drop by drop. During the process solution was mixed vigorously. Solution was heated until color’s change is evident (pale yellow). Then it was removed from the heating element and stirred until cooled to room temperature.

Antibacterial assay

The antibacterial activity of the synthesized AgNps was evaluated by the Nutrient Agar Well Diffusion Assay. The bacterial cultures were grown on the nutrient agar media by sub-culturing them on fresh slants and incubating them at the appropriate temperature for 24 h. A suspension of each test microorganism from slants was prepared using sterile distilled water. After pouring 20 ml of nutrient agar media into each petri plates and left solidifying, 0.5ml of each microorganism suspension was added in petri plates and these were moved in circular motion to spread the suspension on whole nutrient agar surface. Using a borer, approximately 5 mm diameter, wells were bored in these plates and AgNps was added into the wells. The AgNps were used in a concentration/composition as they were synthesized. The plates were incubated at 37°C for 24 h. The antibacterial activity was determined by measuring the diameter of the inhibition zone in mm.

Toxicity determination of silver nanoparticles

All the mice were weighed before and after the process. It was done to determine that how silver nanoparticles affect the weight of mice after being injected for four weeks. Mice were anaesthetized on 29th day of experimental work. They were dissected and their organs were separated. Morphological characters of each organ were noted and compared against the healthy organs of the control mice. Microtomy of organs of albino mice was done and observed under the compound microscope.

Results and Discussion

Characterization of silver nanoparticles

As mentioned earlier, the Silver nanoparticles were synthesized with two different methods namely:

• Starch encapsulation.

• Colloidal synthesis.

These were labeled as ST1 and ST2 respectively. These were characterized through FTIR, SEM and XRD. Many properties and characteristic features of Silver nanoparticles were observed. The results are as follows:

FTIR analysis

FTIR spectra for both samples of Silver nanoparticles (ST1 and ST2) were obtained in the range of 4000-400/cm. This study was done in order to find out the components present in the particular compound i.e. in both samples of synthesized Silver nanoparticles. The distinct peaks were formed and were plotted in the form of graphs. In the following image, a comparison of both of the samples is shown. The spectra of both samples of Silver nanoparticles were studied. A sharp peak was observed in the region around 3500 cm-1 a wide strong peak is observed at 3339 cm-1 which would be due to O-H stretching vibration owing to the interaction of water molecules with silver nanoparticles surface. A sharp peak at 1636 cm-1 and another in the range of 1000-1500 cm-1 represents the presence of NO2 in nanoparticles formed by precursor Silver Nitrate during synthesis of Silver nanoparticles (Figure 1).

pharmacology-toxicological-FTIR-analysis-Silver

Figure 1: FTIR analysis of Silver nanoparticles (ST1 and ST2).

SEM (scanning electron microscope) analysis

SEM analysis of ST1 silver nanoparticles showed that the particles were hexagonal in shape whereas some of these are also found to have rhombhohedral shape i.e. a cubical rhombus with the approximate calculated size between 90-150 nm. ST2 silver nanoparticles were having flaky shape with the calculated size between 80-120 nm. The following image shows the SEM images of Silver nanoparticles on right side whereas on left side of the image, the zoomed in images of synthesized Silver nanoparticles can be seen in Figure 2.

pharmacology-toxicological-ST2-Silver-nanoparticles

Figure 2: SEM analysis of Silver nanoparticles (ST1 and ST2). (a and b) Hexagonal and Rhombohedral shape of ST1 Silver nanoparticles; (c and d) Flaky shape of ST2 Silver nanoparticles.

XRD analysis

Powder XRD of both ST1 and ST2 silver nanoparticles was done. The obtained peaks are shown in the Figure 3, which all were indexed with, which show a typical structure of Silver (Ag). A few weak peaks were also observed. In general, it is observed that nanoparticle size decreases in ST2 as compared to ST1. Above shown is the powder XRD result of ST1 and below is of ST2 Silver nanoparticles. According to the Sherrer equation, D=0.8aλ/Bcosθ, where λ is wave of radiations and θ is diffraction angle.

pharmacology-toxicological-XRD-analysis-Silver

Figure 3: XRD analysis of Silver nanoparticles (ST1 and ST2).

Antibacterial effects of silver nanoparticles

Antibacterial assay was carried out by Liquid method and Plate method. The details are as follows:

Liquid growth method

Antibacterial activity of synthesized Silver nanoparticles was done against gram positive and gram negative bacteria by using liquid growth method.

Against Bacillus subtilis

The results were obtained by taking absorbance at 600 nm after every two hours of incubation for 48 h. Growth curve was plotted between absorbance at 600 nm and time interval in hours (Table 1 and Figure 4).

S. No. Time interval (Hours) Different concentrations of Silver nanoparticles in Bacillus subtilis
Control 5 ml 10 ml 15 ml 20 ml 25 ml
1 0 0.08 0.075 0.078 0.071 0.073 0.079
2 2 0.081 0.078 0.079 0.089 0.077 0.084
3 4 0.08 0.081 0.08 0.085 0.089 0.081
4 6 1.675 0.085 0.088 0.165 0.105 0.104
5 8 1.725 0.099 0.097 0.112 0.103 0.113
6 10 0.899 0.08 0.083 0.098 0.085 0.102

Table 1: Antibacterial effects of Silver nanoparticles by liquid method against Bacillus subtilis.

pharmacology-toxicological-effects-silver-nanoparticles

Figure 4: Antibacterial effects of silver nanoparticles by liquid method against Bacillus subtilis.

Against Escherichia coli

The results were obtained by taking absorbance at 600 nm after every two hours of incubation for 48 h. Growth curve was plotted between absorbance at 600 nm and time interval in hours (Table 2 and Figure 5).

S. No. Time interval (Hours) Different concentrations of Silver nanoparticles in E. coli
Control 5 ml 10 ml 15 ml 20 ml 25 ml
1 0 0.15 0.117 0.154 0.102 0.094 0.096
2 2 0.157 0.118 0.115 0.091 0.08 0.094
3 4 0.279 0.112 0.136 0.101 0.09 0.081
4 6 2.198 0.114 0.112 0.09 0.083 0.091
5 8 2.221 0.147 0.17 0.134 0.112 0.095
6 10 2.182 0.104 0.11 0.098 0.088 0.061

Table 2: Antibacterial effects of Silver nanoparticles by liquid method against E. coli.

pharmacology-toxicological-Antibacterial-effects-silver

Figure 5: Antibacterial effects of silver nanoparticles by liquid method against Bacillus subtilis.

Plate method

The antibacterial assay was also carried out with Plate method. The nutrient agar media was solidified and bacteria were grown. The zones of inhibition were calculated against ST1 and ST2. The results are shown in Figure 6.

pharmacology-toxicological-Plate-method-ST1

Figure 6: (a) Plate method for ST1 against E. coli; (b) Plate method for ST2 against B. subtilis; (c) Plate method for ST2 against E. coli; and (d) Plate method for ST2 against E. coli.

The results of antibacterial assay carried through plate method are shown in the tables and respective data is also shown in the form of Figures 7 and 8. Tables 3-5 and Figure 3 show the antibacterial activity against Bacillus subtilis. Table 4 and graph 4.4 shows the antibacterial activity against E. coli.

Ag Np Zone of inhibition (mm)
ST1 2.9
ST2 1.6

Table 3: Zone of inhibition (mm) produced by ST1 and ST2 against Bacillus subtilis.

Ag Np Zone of inhibition (mm)
ST1 2.6
ST2 1.9

Table 4: Zone of inhibition (mm) produced by ST1 and ST2 against E. coli weighing of organs of mice after dissection.

Data of Control set of mice.
S. No. Organs of mice Wt. in grams after 28 day experiment
  Mice 1 Mice 2
1 Intestines 1.54 1.69
2 Liver 0.79 0.82
3 Spleen 0.14 0.13
4 Stomach 0.18 0.19
5 Kidneys 0.21 0.2

Table 5: A comparison of weights or organs of control mice after 28 days.

pharmacology-toxicological-against-Bacillus-subtilis

Figure 7: Zone of inhibition (mm) produced by ST1 and ST2 against Bacillus subtilis.

pharmacology-toxicological-Zone-inhibition

Figure 8: Zone of inhibition (mm) produced by ST1 and ST2 against E. coli.

Determination of toxicity of silver nanoparticles

The protocol followed to study the in vivo toxicity in mice was based on a 4 week study. Few morphological characters were noted and then the Microtomy was done in order to study the toxicity caused in the organs of mice. The results obtained and the data gathered (Tables 5-7 and Figures 9-12).

Data of mice treated with Ag-Nps (ST1).
S. No. Organs of mice Wt. in grams after 28 day experiment
  Mice 1 Mice 2
1 Intestines 1.65 2.12
2 Liver 0.81 1.32
3 Spleen 0.12 0.14
4 Stomach 0.19 0.15
5 Kidneys 0.2 0.23

Table 6: A comparison of weights or organs of mice treated with ST1 after 28 day.

Data of mice treated with Ag-Nps (ST2).
S. No. Organs of mice Wt.(in grams) after 28 day experiment
  Mice 1 Mice 2
1 Intestines 1.52 1.98
2 Liver 0.77 0.81
3 Spleen 0.13 0.15
4 Stomach 0.19 0.18
5 Kidneys 0.22 0.21

Table 7: A comparison of weights or organs of mice treated with ST2 after 28 days.

pharmacology-toxicological-Male-albino-mice

Figure 9: (a) Male albino mice in cage; (b) Male albino mice before dissection; (c) Albino mice after dissection; and (d) Albino mice after removal of organs.

pharmacology-toxicological-comparison-weights-organs

Figure 10: A comparison of weights or organs of control mice after 28 days.

pharmacology-toxicological-organs-mice-treated

Figure 11: A comparison of weights or organs of mice treated with ST1 after 28 days.

pharmacology-toxicological-comparison-weights-organs

Figure 12: A comparison of weights or organs of mice treated with ST2 after 28 days.

Microtomy

The organs were subjected to the procedure of Microtomy. The slides were prepared and were observed under the microscope. The black patches on the sections of the following figures indicate the accumulation of Silver nanoparticles in the organs of male albino mice (Figure 13).

pharmacology-toxicological-Various-pictures-organs

Figure 13: Various pictures of organs of male albino mice observed under microscope.

Discussion

In this particular research, silver nanoparticles were synthesized with two different methods.

• Starch encapsulation.

• Colloidal method.

It was done in order to synthesize the silver nanoparticles in two different sizes so that the size based study can be carried out in an efficient and appropriate way.

The First half explains the antimicrobial effects of Silver nanoparticles. The antibacterial assay was carried out with two bacterial strains, Escherichia coli and Bacillus subtilis.

Two methods were opted:

• Liquid method.

• Plate method.

Nutrient Broth and Nutrient agar were used in the two methods respectively. Bacterial cultures were kept in slants at optimum temperature. These were spread on the plates with the help of inoculating needle and were subjected to ST1 and ST2 and were kept for 24 h in an incubator. Readings of triplicates were taken after 1 day in millimeters and the mean value was taken. The data was kept in tabular form and graphs were made accordingly. All the results depicted that both ST1 and ST2 has the potential to kill the microbes.

The second half of this research includes the toxicity that can be caused by the intake of silver nanoparticles. It is of no doubt that silver nanoparticles can have unlimited benefits, yet, it might can pose a life threatening harm to humans. In this particular study, 6 male mice were brought from VRI (Veterinary Research Institute, Lahore cantonment). These were of 4 weeks of age. Their acclamation period was one week. They were weighed and results were noted. After to it, the experimental procedure started. They were intravenously injected by silver nanoparticles for consecutive 28 days. 2 mice were kept as control. 2 were injected with ST1 in low and high doses. 2 mice were injected with ST2 in low and high doses. Morphological characters were observed. The mice with the high doses of ST1 and ST2 seem to have slight changes in their coat colors (fur was turned into tan color). They were again weighed on 29th day and results were noted. Later, they were anesthetized and dissected. Their organs were separated and were weighed for comparison studies. Spleen seems to have the increased size in the experimental mice as compared to the healthy mice. Later, microtomy for the organs of mice was done. Here the fine sections of the organs were made and slides were prepared to be observed under the microscope. The results showed the black pigmentation within the tissues of the organs. It shows that the intake of silver nanoparticles might cause organ toxicity in mice.

From the above results and discussions, it is clear that Silver nanoparticles have antibacterial properties and it may cause organ toxicity in mice. On daily basis, crime scene investigators come up with different types of cases that can be best related to the cause of death. Few of such cases are related to death caused by poisoning and slow poisoning. Silver nitrate or their counterparts can be injected to some person intentionally to cause organ toxicity in humans that may result to cause the death of the person. This research was carried out in order to find out that to which extent Silver nanoparticles can be toxic to mice. These results can be applied to humans as well. As nanotechnology have many applications worldwide. One of it is in Forensics. This research data can be helpful in finding out whether a person is died due to natural causes or is being poisoned through Silver nanoparticles. After post-mortem, the sections of the tissues can be cut and observed under microscope and it can reveal that the person was intentionally poisoned to death. So, this way, the mystery of a dead person can be solved in an efficient manner.

Conclusion

Among nanoparticles, silver nanoparticles are getting much attention in almost every field. In this study, I figured out that Silver nanoparticles have antibacterial properties and significant results were noted. Moreover, the toxicity caused by silver nanoparticles was also observed in an in vivo study on albino mice. The mice were injected with a proper low and high dose for 28 days. The mice showed significant results in toxicity. So, this data explained, the toxicity and antibacterial properties can be useful in Forensics and in pharmacological industry to solve mystery of death and for the production of novel antidote against these toxic substances.

Acknowledgement

The authors would like to acknowledge the Bio-process laboratory, Department of Chemistry, GC University Lahore for providing thebacterial strains, The Veterinary Research Institute, (VRI) Lahore Cantonment and Punjab forensic Science Agency for providing the facilities of SEM-EDX.

References