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HIGH SCHOOL NANOSCIENCE PROGRAM

Biotoxicity

 

Background

Figure 1. Silver nanoparticle synthesis.  A. Silver nitrate provides Ag+ ions that are reduced by sodium citrate, which itself becomes oxidized. Excess sodium citrate present in the solution caps the particles through the carboxylate groups and prevents them from growing and aggregating as larger particles; this procedure is carried out at elevated temperature to decompose the citrate and increase the speed of particle growth. The ratio of sodium citrate to silver ions causes a change in nanoparticle size.   B. Transmission electron micrograph of the silver nanoparticles and a higher magnification image showing the crystalline planes, indicating that these are nanocrystals of silver.
Figure 2. Increase in surface area with decreasing particle size.

Nanoparticles are particles of any material that are less than 100 nm in diameter.  On the nanoscale, material properties can change from those observed in the bulk.  One easily observable example is the color. Bulk silver is the typical shiny silver color we are accustomed to, but silver nanoparticles can be a variety of colors, depending on the shape and size.  Particles that are approximately spherical and below 20 nm in diameter will display a clear yellow or amber color.   These absorb light very strongly; in fact, a very dilute solution can display a bright color.  

Before the 20th century, the only known method of processing silver was by grinding and reducing it to fine powder. This presented a problem since the fine silver powder does not stay suspended in the solution for long.  In nanoparticulate form, they can be suspended in a liquid base, which allows for solution processing of tiny, crystalline metallic particles (Figure 1).  Also, the surface area greatly increases, allowing access to the majority of silver atoms for increased reactivity, as demonstrated in Figure 2.  For example, U.S. silver dollar was once placed in milk jugs for prevention of microbe growth.  This coin contains 26.96 grams of coin silver, has a diameter of about 40 mm, and has a total surface area of approximately 27.70 square centimeters. If the same amount of coin silver were divided into particles 1 nanometer in diameter – the total surface area of those particles would be 11,400 square meters, which is 4.115 million times greater than the surface area of the silver dollar and much more effective as antimicrobials!

It is well known that silver ion and silver-based compounds are highly toxic to microorganisms, showing strong biocidal effects against as many as 16 species of bacteria, including Escherichia coli, and fungi. The mechanism of silver toxicity relies on the interaction between the metal and cellular membranes. At the plasma membrane, silver binds either to membrane bound proteins or the lipid bilayer itself and destabilizes the membrane, causing ion leakage and cell rupture. Inside the cell, silver binds to and disrupts the function of mitochondrial membranes, interfering with the energy (ATP) yielding reactions of the respiratory chain. Silver has also been shown to bind specifically to cellular enzymes and DNA and interfere with their functions. Silver ions have only limited usefulness as antimicrobial agents, because large amounts of silver ions are needed, which will also be toxic to the host. Silver nanoparticles can insert within the yeast membrane due to their small size, which may destabilize the membrane, and release silver ions locally from the silver particle directly into the microbe. The antimicrobial mechanism of continuous release of silver ion from the metallic nanoparticulate form can overcome the limitation of biotoxicity to the host as much smaller amounts are needed. The mode of action of nanoparticulate silver is thought to be similar to that of silver ions. However, due to either disruption of the membrane and/or local release of silver ions, the effective concentration of nanoparticles is much less than in silver ions.

The biotoxic activity of silver nanoparticles is related to their size, with the smaller particles having higher activities when normalized for silver mass content. For example, it is known that smaller silver nanoparticles are more effective in killing E. coli bacteria than large ones, and typical diameters of less than 10 nm are the best in this regard.  In this experiment, silver nanoparticles are stabilized from aggregation by adsorption of surface capping groups in the form of citrate (Figure 1a).  Once silver nanoparticles aggregate, significant loss of the antibacterial activities occurs due to their inability to penetrate the plasma membrane and loss of surface area. The synthetic method used has a larger than 1:1 molar ratio of sodium citrate to silver, which is necessary for growth and stabilization.  A portion of the citrate is used to reduce the silver ions to silver metal, while the remaining citrate caps and protects the particles from aggregation.  **Important** The size of the silver nanoparticles produced is very sensitive to the temperature and reaction time.  Expect variations in size for each synthesis.  The smallest nanoparticles are a clear yellow color, while increases in size lead to amber, brown, and finally a cloudy brownish/green color, which are increasingly less effective as antimicrobial agents.  Note the color of the particles before addition to the yeast and watch for trends in biotoxicity.

This experiment measures the rate of respiration of yeast in the presence of different forms of silver. The yeast used is Saccharomyces cerevisiae (Budding, bread, or bakers yeast). It is commonly used as a model organism for eukaryotic biology because many of the cellular functions in found yeast are conserved in human cells. This lab uses Saccaromyces cerevisiae as a model for infectious fungi such as Candida albicans. The experiment uses a change in the rate of respiration to measure toxicity of silver nanoparticles. Respiration is the process by which a cell converts glucose (or other sugars) and oxygen into cellular energy (ATP), carbon dioxide and water. In eukaryotes, this process occurs in a membrane bound organelle called mitochondria. The outer membrane of mitochondria contain a large number of membrane bound proteins that allow for the movement of molecules and ions. The inner membrane contains a specific complex of membrane proteins that participate in respiration. The movement of molecules and proper function of these membrane proteins is critical to ATP production and respiration. Silver nanoparticles disrupt this function.

Figure 3 (left). Representative nanoparticle syntheses:   A.) remove from water bath while pale yellow;    B.) as the particles cool, they will continue to react and turn yellow.   C.) If reaction occurs too long, particles will continue to grow and become cloudy/brown and these are less effective as antimicrobials.

The experimental setup in Figure 4 shows the method for collection of gas in an inverted graduated cylinder by displacement of water.

Figure 4: Experimental Set-up. Flask contains yeast and sugar only (A) or with additional silver nanoparticles (B), silver powder (C) or silver nitrate (D).

Historical uses of silver as an antimicrobial.
The earliest record of silver as an antimicrobial comes from Herodotus in 450 BC who told of the King of Persia keeping boiled water in flagons of silver to keep the water fresh. Ancient Romans would keep silver pieces in the bottom of milk containers. In the 17th and 18th centuries, silver nitrate was used to treat open wounds (especially due to burns). It was shown in 1869, that Aspergillus niger could not grow in silver lined vessels. In 1880, a silver nitrate solution was used to prevent opthalmia neonatorum, a common eye infection in newborns, and a similar treatment is still used to this day. In the late 1940s, silver sulphadiazine was used as the standard treatment for burns. It offered general antimicrobial properties with out some of the side effects of antibiotics.

Current Uses of Silver Nanoparticles as Antimicrobials.
Silver and silver nanoparticles are found in a wide variety of products to take advantage of silver’s antimicrobial properties. Curad and Band-Aid sell bandages coated with silver colloids. In common medical practice, silver is used as aseptic cover for plastic surgery, traumatic wounds, leg ulcers, skin grafts, incisions, abrasions and minor cuts. It is used to coat catheters and wound bandages. Outside of the medical field, silver is used in institutional water distribution systems and can be found in Brita water purification systems. Silver is used to sterilize recycled drinking water aboard the Russian MIR space station and on the NASA space shuttle. In Japan, silver is mixed into plastics for antimicrobial protection of telephones, calculators, toilet seats, children’s toys, and pacifiers. Silver can also be found imbedded into sports fabric, sleeping bags, and sports socks.


Figure 5 (left): Ionic silver starts killing a broad spectrum of pathogens within 30 minutes of exposure to the dressing as demonstrated by in vitro testing, including aerobic and anaerobic bacteria.

Figure 6 (right): ACTICOAT Moisture Control is an absorbent antimicrobial dressing, which offers the antimicrobial protection synonymous with ACTICOAT & SILCRYSTTM nanocrystalline+ silver, in a highly absorbent, easy to use format. The product consists on a silver coated wound contact layer, a highly absorbent foam and a waterproof top film.

Figure 7: Silver-nanoparticle-embedded antimicrobial paints on glass slides without coating (A), glass slides coated with paint only (B) and glass slides coated with silver nanoparticle-embedded paint (C), onto which E. coli cells were sprayed and incubated at 37 °C overnight. Each black dot corresponds to a bacterial colony grown from a single surviving bacterial cell. Note lack of growth (dots) on nanoparticle-embedded paint (c). (Nature Materials 7, 236 - 241 (2008))