Examining the cytotoxicity of metallic nanoparticles…a little blast from the past.

This is just a little experiment I did with a classmate during my undergraduate years to see if metallic nanoparticles could have an effect on bacteria and yeast, which could perhaps be used to address the use of nanoparticles in a microbicidal capacity.

Here goes the report.

Abstract

Introduction

An investigation was carried out on the cytotoxic properties of metallic nanoparticles on both Prokaryotic and Eukaryotic model organisms using qualitative assays for cytotoxicity.

Methodology & Results.

Metallic nanoparticles of Copper, Silver and Iron were prepared using Turkevich synthesis, these were then screened for cytotoxicity using well diffusion assays using a reference set of four bacterial species, results indicated that Silver nanoparticles were potently cytotoxic against bacteria, being capable of inducing substantial zones of inhibition in the medium into which said particles could
diffuse.

The test was repeated for Eukaryotes using Saccharomyces cerevisiae as the model organism and turbidimetry for evaluation. Values were obtained as a function of varying concentration vs varying turbidity. Results indicated a direct statistical relationship between the strength of nanoparticle
solutions of Silver as opposed to the other two.

Conclusions
The cytotoxicity of nanoparticles of silver was demonstrated in both prokaryotes and eukaryotes, this backed up the previously reported instances of nanosilver toxicity in the scientific literature & highlighted the potential concerns associated with environmental contamination of the environment
with metallic nanoparticles. It also raised questions about the potential use of silver nanoparticles in conjugates as a form of cytotoxic therapy, which may be worth investigating.

Materials and Methods

Turkevich Synthesis

Materials Required

0.1 M Silver Nitrate solution, 0.1 M Copper Sulphate Solution, 0.1 M Ferrous sulphate solution, 38.8 M Sodium Citrate solution (aqueous) , deionized water, standard glassware.

Principle

Turkevich synthesis uses a reducing agent to first reduce metal salts to nanoparticles, the same agent then inhibits re-coagulation.

Procedure

Solutions of the aforementioned nature were prepared from raw salts using deionized water, then reaction mixtures were prepared using metal salt solutions and sodium citrate in the ratio 2:1.

A water bath was heated to 100’C and the reaction mixtures were boiled until colour changed , this change in colour is often attributed to the Surface Plasmon Effect, which is due to changes in the dielectric properties of synthesized nanoparticles.

The solutions, once colour had changed, were gradually cooled back to room temperature and stored in reagent bottles, estimation of the concentrations of the nanoparticles and shape-size characterization weren’t carried out due to the lack of access to a Scanning Electron Microscope.

The production of nanoparticles was verified by cross-corroborated change in colour due to the surface plasmon effect.

Assay for Prokaryotic Cytotoxicity

Materials Required

Bacterial broth cultures, 24-48 hours old , of Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes and Escherichia coli, cork borer, nutrient agar/blood agar base medium, laminar airflow unit, swabs, micropipettes and pipette tips, Ethanol (70%), standard glass labware.

Procedure

All non-heat labile materials were sterilized by autoclaving under standard conditions, 121’C at 15psi, 20 minutes, heat labile materials such as pipettes were sterilized using ultraviolet irradiation. Sterile, aseptic conditions were established for carrying out the experiment

Swab cultures were carried out and a well diffusion assay was set-up using 75 microliters of the nanoparticle dispersions in the first round and a much higher concentration of 200 microliters in the second phase of the experiment, in the preliminary, first round, just E.coli and S.aureus were used, and the well diffusion assay took into account dilutions of nanoparticles (x, x/10 & x/100) , it was found that preliminary antimicrobial activity of any significant potency was only shown by the dispersions at their original concentrations, therefore this was the only concentration chosen for further corroborative investigations.

Results of prokaryotic cytotoxicity assay. Zones of inhibition are clearly apparent.

The process was repeated the second time with all four of the aforementioned cultures, with only original concentrations of the nanoparticle dispersions being used.

Nansosilver at the original concentration showed the maximum amount of microbicidal activity, which by inference makes it cytotoxic to prokaryotes, data about ZOI diameters is available in the appendix that follows this report.

Assay for Eukaryotic Cytotoxicity

While the initial plans included the use of trypan blue cell exclusion method for corroboration, the inconsistency of the yeast dispersion used meant that the results were practically unusable, and therefore data had to be tentatively evaluated using turbidimetry alone, we recommend caution before jumping to conclusions on the basis of this experiment alone and suggest further corroboration and verification.

Materials

yeast dispersion, 1 pellet in 50 ml of sucrose broth (approximately 0.5 g added) , nanoparticle dispersions with dilutions (x , x/10 and x/100) respectively, photocolorimeter (a spectrophotometer was preferable but not available)

Procedure.

Control and test solutions were created for each set of nanoparticle dispersions, for all three metallic nanoparticles, the controls consisted of 1 ml of the nanoparticle dispersions, and 1 ml of uninoculated sucrose broth and 8 ml distilled water, while the test samples contained 8 ml distilled water, 1 ml nanoparticle solutions and 1 ml of inoculated sucrose broth, the tubes were plugged with cotton and incubated overnight at room temperature (25-27’C)

Readings for optical density were then taken after 24 hours of culture, to examine if there was a trend between dilutions and rate of cell growth, a direct relationship between the two variables would yield an inversely proportional relationship.

A trend of cytotoxicity was found to occur for treatment with nanosilver solutions, this has been corroborated by studies which have illustrated the ability of silver nanoparticles to induce mutations and cell death (P V Asharani et al 2008 Nanotechnology) The other two nanoparticle dispersions showed no cytotoxicity.

The raw data and the graphical analysis of the data from this experiment is also available in the appendix at the end, a baseline correction factor was included to make presenting information in the graphs easier, the correction was applied by adding an equal amount to all derived results.

Results and Conclusions

Turkevich synthesis can be carried out in simple facilities to produce nanoparticle dispersions for further investigation.

Silver nanoparticles are potently cytotoxic against all the organisms tested against in the prokaryote cytotoxicity assay, regardless of whether they are gram +ve or gram –ve, and as such can be used as microbicides.

However, silver nanoparticles have been shown to be cytotoxic against Eukaryotes too, this means that unregulated efflux into the environment can lead to disastrous ecological consequences, and therefore strict regulatory norms are imperative.This cytotoxicity, however could have biomedical applications in the form of immunoconjugates against cancer specific receptors, for instance,

The other nanoparticle dispersions (Copper and Iron) , at the tested concentrations, were found not to be cytotoxic , as were silver dispersions at less than x/10 dilution, this could indicate a preliminary statistic for hazard levels and an association with the concentration, while clearing the other two for free usage at the investigated concentrations.

However, for the data to become fully valid for policymaking, an accurate method such as mass spectrometry must be used to evaluate precisely the concentration of the nanoparticles in their respective dispersions.

To sum up, our data is in agreement with research findings that silver is cytotoxic, and these studies so far point towards iron and copper not being cytotoxic, however, further research needs to be carried to account for any imperfections in the study itself; these results are a rough starting point, at best.

References

P.K. Khanna, Narendra Singh, Deepti Kulkarni, S. Deshmukh, Shobhit Charan, P.V. Adhyapak, Water based simple synthesis of re-dispersible silver nano-particles.

Iron nanoparticles: Synthesis and Applications in surface enhanced Raman Scattering and electrocatalysis, Guo et al, Physical Chemistry Chemical Physics, 2001.

An antibiotic assay by agar well diffusion method, Perez et al, Acta Biol. Med. Exp, 1990

Trypan Blue Exclusion Test of Cell Viability , Strober, Current Protocols in Immunology,

Toxicity of silver nanoparticles in zebrafish models
P V Asharani et al 2008 Nanotechnology 19 255102 (8pp) doi: 10.1088/0957-4484/19/25/255102

Appendix A : Turbidimetry Data and Graphs

Results from turbidimetry experiment.

Appendix B : Prokaryotic Cytotoxicity Assay.

Prokaryote zone of inhibition data.

Appendix C: Tentative estimates of concentration.

Silver Nanoparticle dispersion.

100 ml of AgNO3 solution contained 10.8 mg of Silver
3 ml of the finally prepared solution contained 0.324 mg of Silver nanoparticles

Solutions for the Yeast assay, contained 0.108 mg (x) , 0.0108 mg (x/10) and 0.00108 mg (x/100) of
Silver & 200 Microlitre aliquots, used for the prokaryote assay, contained 0.0216 mg of silver

Iron solution.

100 ml of FeSO4 solution contained 5.6 mg of Iron

3 ml of the prepared nanosolution contained 0.168 mg of Iron Nanoparticles
1 ml, for the yeast assay, contained 0.056 mg (x) , 0.0056 mg (x/10) , 0.00056 (x/100)

200 Microlitre aliquots, for prokaryote assays, contained 0.0112 mg of Iron nanoparticles

 

Copper solution.

100 ml of CuSO4 solution contained 6.35 mg

1 ml of the prepared solution contained 0.0635 mg (yeast assay) , the other two quantities used were 0.00635 mg and 0.000635 mg.

200 Microlitre aliquots contained 0.0127 mg

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2 responses to “Examining the cytotoxicity of metallic nanoparticles…a little blast from the past.

  1. hello ankur,i have just started working on silver nanoparticles. while performing well diffusion assay for antimicrobial activity,every time when i perform it,the nanoparticles get deposited at the bottom of the well as black residue,can you suggest me something to rectify it. i use LB agar(2% agar).

    • Hi Armendra.

      What form are you using the nanoparticles in? I had mine suspended in sodium citrate.
      The agar doesn’t appear to be a problem – it appears your particles are falling out of suspension for some reason – you will need to
      try various buffers/carriers that are inert by themselves. If that doesn’t work and if you have access to a rotary shaker you can just
      keep the particles dispersed in LB broth and measure impact on growth by turbidimetry.

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