Instructor Information Silver Nanoparticles: Antimicrobial Fact, Fiction, or Pandora’s Box?
Developed by: Jon Pazol, West Leyden High School Graduate Student Mentor: Ann Czyzewski Faculty Advisor: Annelise Barron
Table of Contents
Teachers Guide & Background Information ...................................... 3 Goals and Objectives ........................................................................ 5 Standards.......................................................................................... 6 Material List....................................................................................... 7 Lesson / Module Development.......................................................... 8 Lesson / Module................................................................................ 11 Appendix (Research Background) ................................................... 19
Teachers Guide & Background Information
Silver nanoparticles have the potential to revolutionize the medical and consumer products industries. These particles, which range in diameter from 4-20 nanometers , demonstrate unique properties, especially against various bacteria, viruses, and fungi. Silver has been used to treat infections for centuries, but with the advent of nanotechnology, the use of silver in nanoparticle form has opened new treatment avenues. Various researchers have found that silver nanoparticles are effective killers of pathogenic bacteria such as E.coli, B. subtilis, and S. aureus. The antimicrobial mechanisms of their action are not completely understood, but they may interact with the bacterial peptidoglycan layer, form pits in the cell wall, change membrane polarity, and/or form free radicals that damage the membrane, or a combination of these effects. One thing that is known is that because the nanoparticles do not act via cell receptors, there is no immune response and thus no antibacterial resistance. Recent studies have also indicated that silver nanoparticles in the range of 1-10 nm attach to HIV proteins and inhibit the virus from binding to cells. Because of these antimicrobial properties, silver nanoparticle technology has been incorporated into surgical instruments, hospital wound dressings, and a wide variety of consumer products, such as washing machines, food storage containers, bandages, and clothing. In 2005 and 2006 The Sharper Image introduced FresherLonger™ food storage containers, Curad® began selling its Curad® Silver bandage line, and Champion and Arc Outdoors began marketing clothing incorporating silver nanoparticles into the fabric. The possibilities for using the antimicrobial properties of this technology seemed boundless. Then, in November 2006, the Environmental Protection Agency (EPA) established regulations that, “any company wishing to sell a product that it claims will kill germs by the release of nanotech silver or related technology will first have to provide scientific evidence that the product does not pose an environmental risk” (Washington Post 11/23/2006). In a related Nanotechnology White Paper published in early 2007, the EPA further asserted the steps that companies would have to undergo before they could incorporate nanotechnology into their products and make claims regarding the antimicrobial properties. Consequently, the Sharper Image, Curad®, and Champion C9 have dropped references to silver nanoparticles in their literature and packaging, but they have not changed the materials themselves. Using silver medicinally, usually in the form of colloidal silver sold in health food stores as a dietary supplement, has some potential side effects. These include: argyria – a permanent incorporation of silver into the skin resulting in a silver skin tone, neurologic problems, kidney damage, stomach distress, headaches, fatigue, skin irritation, and the potential interference with the body's absorption of some drugs. The effects of silver nanoparticles on the soil, air, wastewater, groundwater, and bacteria, algae, protozoa, fungi, plant, and animal life are not at all understood. Consequently, the technology is not without some huge implications, and its further use may result in the opening of a “Pandora’s Box” of problems. During this module, students will understand how silver nanoparticles affect the growth of microorganisms, will set-up controlled experiments that test the antimicrobial
properties of Curad® Silver bandages, FresherLonger ™ food storage containers, and Champion C9® athletic clothing, and will consider the potential environmental impacts of the technology. Through designing their own controlled experiment the students will test the antimicrobial claims of these products as compared to non-silver nanoparticle products. They will also be presented with some of the controversy surrounding the use of silver nanoparticles and will consider the Environmental Protection Agency’s decision to regulate their use, as well as the manufacturer’s response. References •ARC Outdoors Launches E47™ Anti-Odor Fabrics and Yarns. Press Release. 7/26/2005. •Cho, KH., Park, JE., Osaka, T., Park, SG. The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochimica Acta. 2005; 51(5):956-60. •Elechiguerra, J.L., Burt, J.L., et al. Interaction of silver nanoparticles with HIV-I. Journal of Nanobiotechnology. 2005;3(6): •Kim, J.S. et al. Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine. 2007; 3(1):95-101. •National Institutes of Health. 2006. National Center for Complementary and Alternative Medicine. Colloidal Silver Products. NCCAM Publication No. D209. •Sharper Image Introduces FresherLonger™ Miracle Food Storage Containers. Business Wire. March 08, 2006 •Sondi, I. and Salopek-Sondi, B. Silver nanoparticles as antimicrobial agent: a case study on E.coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science. 2004; 275 (1):177-182. •U.S. Environmental Protection Agency. 2007. Science Policy Council. Nanotechnology White Paper. EPA 100/B-07/001 •Weiss, R. EPA to regulate nanoproducts sold as germ-killing. Washington Post. November 23, 2006. •Yang, Y. and Balcarcel, R. 96-well plate assay for sublethal metabolic activity. ASSAY and Drug Development Technologies. 2004; 2(4): 353-61. •Yoon, KY. Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles. Science of The Total Environment. 2007; 373(2-3): 572-75.
Goals and Objectives
• Goal: o o o Understand nanotechnology applications. Objective: Describe the uses of silver nanoparticles. Objective: Understand how silver nanoparticles affect microorganism growth.
Goal: Gain practice in the scientific method. o Objective: Analyze consumer product claims. o Objective: Design controlled experiments to test consumer products. Goal: o o o Consider science, technology, and society issues. Objective: Understand the potential drawbacks of silver nanoparticle technology. Objective: Formulate a position on the use of silver nanoparticles.
State Standards: This activity meets the following Illinois Learning Standards for Science as presented by the Illinois State Board of Education for Early and Late High School1
STATE GOAL 11: Understand the processes of scientific inquiry and technological design to investigate questions, conduct experiments and solve problems. A. Know and apply the concepts, principles and processes of scientific inquiry. 11.A.4a Formulate hypotheses referencing prior research and knowledge. 11.A.4b Conduct controlled experiments or simulations to test hypotheses. 11.A.4c Collect, organize and analyze data accurately and precisely. 11.A.4e Formulate alternate hypotheses to explain unexpected results. 11.A.4a Formulate hypotheses referencing prior research and knowledge. 11.A.5b Design procedures to test the selected hypotheses. 11.A.5c Conduct systematic controlled experiments to test the selected hypotheses STATE GOAL 12: Understand the fundamental concepts, principles and interconnections of the life, physical and earth/space sciences. A. Know and apply concepts that explain how living things function, adapt and change. 12.A.4b Describe the structures and organization of cells and tissues that underlie basic life functions including nutrition, respiration, cellular transport, biosynthesis and reproduction.
STATE GOAL 13: Understand the relationships among science, technology and society in historical and contemporary contexts. A. Know and apply the accepted practices of science. 13.A.4a Estimate and suggest ways to reduce the degree of risk involved in science activities. 13.A.4b Analyze the validity of scientific data by analyzing the results, sample set, sample size, similar previous experimentation, possible misrepresentation of data presented and potential sources of error. 13.A.4c Describe how scientific knowledge, explanations and technological designs may change with new information over time (e.g., the understanding of DNA, the design of computers).
B. Know and apply concepts that describe the interaction between science, technology, and B. society. 13.B.4e Evaluate claims derived from purported scientific studies used in advertising and marketing strategies.
•Nutrient agar (Ward’s 88 V 1500) or tryptic soy agar (Ward’s 88 V 1815) –in pkg. 6 microwavable bottles – 1 bottle pours 10-15 plates •Serratia marcescens D1 (Ward’s 88 V 0997) or Kocuria rosa (Ward’s 88 V 0950) or bean infusion culture (dried beans in water left at room temperature for 48 hrs.) or other suitable bacteria culture •Petri dishes, sterile – 100 x 15mm (VWR 25384-342) •Disposable transfer pipets (VWR 16001-174) •FresherLonger ™ food storage containers (Sharper Image) •Rubbermaid/Tupperware/Store brand food storage containers (Various) •Curad®Silver and Curad “regular” bandages (Osco Drug, CVS, Walgreen’s) •Champion C9 antimicrobial* - “Training” shirts and “Comfort Stretch Compression socks and non-antimicrobial clothing – “Active Performance” shirts and noncompression socks (Target) or Arc Outdoors X-System E47and non X-System clothing (www.arcoutdoors for dealer list) •Colloidal silver solution – particles measured in nm(Vitamin Shoppe, GNC, etc.) •Strawberries, blueberries, raspberries (Various) •Cotton swabs, Ziplock bags, and gauze (Various) •Forceps (Ward’s 14 V1001) •Incubator (Ward’s 15 V 0600) – use will depend on recommended incubation of bacterial culture. *According to Champion and Haynes – these products may or may not contain silver nanoparticles.
Pre-Day 1: Set-up 1. Start bacterial culture according to directions or start bean infusion Bean infusion - dried beans placed in beaker and left exposed for 24-48 hours. Cloudy/foamy appearance signifies bacterial growth. Culture should be subcultured/diluted every few days. Caution: It will smell after a few days.
2. Pour agar plates – microwavable nutrient agar. 1 bottle = 10-15 plates. Store upside down in refrigerator for 1-2 weeks. Bring to room temperature before adding bacteria. 3. Cut bandages and clothing into 1 cm squares. The clothing squares can be washed and re-used. Day 1-2: Nanotechnology Intro – 1 – 1.5 periods 1. Introduce the concept of nanoparticles and silver nanoparticles. a. Show images – Google image search or www.nanosilver.com b. Show scale - http://www.purestcolloids.com/mesoworld.htm c. Describe how silver nanoparticles are antimicrobial. Show products that incorporate silver nanoparticles. a. Show FresherLonger™ food storage containers. b. Show Champion C9 (or other) antimicrobial clothing. Show commercial at http://www.e47nano.com c. Show Curad® Silver bandages. d. Pass out and discuss “Do Silver Nanoparticle Products Work?” handout.
3. Initiate “Proof of Concept” demonstrations – note: these may be done on Day 1 or started earlier and the results shown on this day a. Food Storage - Take 4-5 strawberries or raspberries, etc. and place them into a FresherLonger ™ food storage container. Place in refrigerator for 7-10 days. Record results. b. Antimicrobial Clothing - Have a volunteer wear a C9 Antimicrobial shirt and C9 Antimicrobial socks during a 1 hour intense aerobic exercise session. Turn the clothing inside-out and place the shirt and socks into separate, sealed ziplock bags for at least 1 week. Record odor results. c. Bandages - Using a transfer pipet, place bacteria broth on the agar side of the petri dish and use cotton swabs to swirl the liquid around so that it covers the entire surface. Place a Curad®Silver square onto the agar. Gently tap into place with forceps. Place the dish upside down into the incubator at the assigned temperature for 24
hours. After 24 hours, measure and record the zone of inhibition of the bacteria colonies around the bandage.
Day 2-3: Experimental Design – Can be done before or after Proof of Concept demonstrations are finished. 1. Discuss/Review the scientific method and steps of controlled experiment a. Hand out “Experimental Design” worksheet b. Approve experimental designs. (Hypothetical student designs below) c. Help students collect materials. 2. Conduct Experiments 3. Collect Data and share with class.
Potential Student Designs Food Storage •Take a pipet of bacteria culture and place into a FresherLonger ™ food storage container, a Rubbermaid locking food storage container, and a generic food storage container. •Place in incubator at recommended temperature for 24 hours. •Take swabs and place on petri dish. Record bacterial growth. Antimicrobial Clothing •Have a volunteer wear a C9 Antimicrobial shirt, 1 C9 Antimicrobial sock, and 1 C9 Nonantimicrobial sock. •Have a different volunteer wear a C9 Non-antimicrobial shirt, 1 C9 Antimicrobial sock, and 1 C9 Non-antimicrobial sock •Wear the clothing during a 1-hour intense aerobic exercise session. •Turn the clothing inside-out and place the shirt(s) and each of the socks into separate, sealed ziplock bags for at least 1 week. •Take swabs and place on petri dish. Record bacterial growth. or •Take 1 cm squares of the various clothing types and place on bacteria filled agar plate. •Place in incubator at recommended temperature for 24 hours. Record zone of inhibition Bandages •Take 1 cm squares of the various bandage types (Curad® Silver, regular, gauze dipped in colloidal silver, etc.) and place on bacteria filled agar plate.
•Place in incubator at recommended temperature for 24 hours. Record zone of inhibition
Day 4: Silver Nanotechnology – Panacea or Pandora’s Box? 1. Examine Pros and Cons of silver nanoparticle use a. Have students research medical and environmental pros and cons of the uses of silver/silver nanoparticles. b. Fill out “Silver Nanoparticle – Panacea or Pandora’s Box” graphic organizer. c. Distribute “EPA Regulates Silver Nanoparticles” and discuss. 2. Have students write letters to the companies, the EPA, newspaper editors, etc
Lesson Worksheet #1
Do Silver Nanoparticle Products Work?
Now that you have been introduced to silver nanoparticles and have seen some of the products that incorporate them, here is what various companies have to say about the technology.
“FresherLonger containers are infused with silver nanoparticles because silver in microscopic particle form is a safe, medically proven antibacterial agent that fights the growth of mold and fungus. In tests comparing FresherLonger to conventional containers, the 24-hour growth of bacteria inside FresherLonger containers was reduced by over 98 percent.” Sharper Image Press Release 3/8/2006 “E47’s anti-odor properties are generated by nanoscale-engineered silver-bearing nanoparticles which permanently bond to specific target fibers at the molecular level. The antimicrobial functionality of E47 fibers is uniformly distributed throughout the fiber and specifically engineered not to flake off, rub off, or wash out – leaving all the other properties of the fiber unchanged.” ARC Outdoors Press Release 7/26/2005
New Curad® Silver Bandages use silver in the wound pad as a natural antibacterial. Laboratory testing showed that silver reduced bacterial growth like Staph. aureaus, E. coli, E. hirae and Pseudomonas aeruginosa-a powerful germ that does not respond to many antibacterials-in the dressing for 24 hours. www.curadusa.com
Based on what you have seen and read, do you believe the companies? Why or why not?
Your teacher is going to conduct some “Proof of Concept” demonstrations with each of the three products. Summarize the process, predict the outcome, and record the results in the chart below: Procedure Prediction Results
Worksheet #2 Experimental Design
Based on the “Proof of Concept” demonstrations, it is your task to test whether or not these silver nanoparticle materials are truly antimicrobial. You are going to design a controlled experiment using agar coated petri dishes and a bacterial culture. The general procedure is as follows: Petri Dish Procedure •Using a marker, number the bottom of a petri dish into ___ sections. •Using a transfer pipet, place bacteria broth on the agar side of the petri dish. •Use cotton swabs to swirl the liquid around so that it covers the entire surface. •Using the pipet, remove any excess and place it into a waste beaker. •Turn the plate upside down so that excess will drip onto the lid. •Place the samples into each of the sections. Use the end of a forceps to gently “tap” the pieces into the agar: •Place the dish upside down into the incubator at the recommended temperature for 24 hours. •After 24 hours, measure and record the zone of inhibition of the bacteria colonies around each of the materials. Experimental Design: Problem: (What you are trying to solve.)
In the space provided, outline your experimental plan in detail. Your experiment must include the following: Hypothesis: (Be specific. It must be able to be tested within the time and resources available.)
(Give exact amounts.)
Procedure: (Be specific. Use step-by-step instructions like a recipe. Record what you did, not what you planned to do.)
Data: (Describe how will it be collected.)
Data table: (Design the table that you will use.)
Worksheet #3 Silver Nanoparticles – Panacea or Pandora’s Box?
A “panacea” is a cure-all or something that will answer many problems. “Pandora’s Box” refers to a mythological story in which someone opened a box and released evils into the world. The use of silver and silver nanoparticles may be seen as both. In the table below, fill out the Pros and Cons of using silver in terms of medicine and the environment.
Worksheet #4 EPA Regulates Silver Nanoparticles
Refer back to the “Do Silver Nanoparticle Products Work?” handout. When the companies first released these products in 2005-06 these press releases “bragged” about the technology. However, late in 2006, the US Environmental Protection Agency (EPA) passed some regulations that restricted their use: “any company wishing to sell a product that it claims will kill germs by the release of nanotech silver or related technology will first have to provide scientific evidence that the product does not pose an environmental risk” (Washington Post 11/23/2006)
In response, some companies changed their advertising. Compare the 2 versions of the product description for FresherLonger™ food storage containers from The Sharper Image: Before the EPA Rulings: “FresherLonger containers are infused with silver nanoparticles because silver in microscopic particle form is a safe, medically proven antibacterial agent that fights the growth of mold and fungus. In tests comparing FresherLonger to conventional containers, the 24-hour growth of bacteria inside FresherLonger containers was reduced by over 98 percent.” Sharper Image Press Release 3/8/2006 After the EPA Rulings: Sharper Image's exclusive FresherLonger™ Miracle Food Storage Containers are made of specially treated air- and odor-impermeable polypropylene and they feature a patentpending, airtight silicone-gasket locking system that helps to retard spoilage. Sharper Image 7/2007
What is the difference between the two descriptions?
Similarly, it is difficult to find silver nanoparticle references for any of the other products. According to company spokespeople, Champion C9 clothing may or may not contain silver nanoparticles, and Curad®, which is regulated by the Food and Drug administration, has also dropped any references. Because the companies dropped references to silver nanoparticles does not necessarily mean that the products no longer contain them. In fact, a loophole in the
EPA’s regulations allow companies to use silver nanoparticles, as long as they do not advertise a connection between them and any antimicrobial properties of the products.
What do you think about this situation?
Do some more independent research about the silver nanoparticle products that you investigated or find other products that currently use the technology. Formulate an opinion about the use of silver nanoparticles. Complete one of the following: 1. Write a letter to a company asking about silver nanoparticle use and environmental hazards. 2. Write a letter to the EPA supporting “closing the loophole” or relaxing the regulations on companies using silver nanoparticles. 4. Write a letter to the editor of a major newspaper explaining silver nanoparticle technology and expressing your beliefs about their pros/cons.
Your letters should include facts/statistics and be written using proper spelling, punctuation, and grammar. ScienceDaily - www.sciencedaily.com and Nano Today www.nanotoday.com offer a variety of articles/information about nanotechnology.
Appendix – Research Background
Enzymatic Assays of Antimicrobial Peptoids on Mammalian Cell Metabolism Jonathan Pazol, Ann Czyzewski, and Annelise Barron Recent news stories have heightened the issue of the increasing number of bacteria that are becoming antibiotic resistant. Scientists are working to develop new classes of antibiotics; however, the bacteria are evolving faster than the introduction of new drugs. Antimicrobial peptides (AMPs) are small (<40 amino acids), naturally occurring proteins found in a variety of organisms that act as antibiotics to which bacteria have not developed resistance. Some of these compounds, such as magainin, which is found in the skin of frogs, would make ideal antibiotics except that they are highly unstable, are easily degraded, may cause adverse immune responses, and are not readily available. Consequently, researchers are working to develop non-natural mimics of these AMPs, and one of the most promising are a group of synthetic peptidomimetic molecules called peptoids. Peptoids differ from proteins in their backbone structure. Typical amino acids consist of a central carbon atom, attached to an amino group, a carboxylic acid group, a hydrogen atom, and a side chain called a replacement group (R) (Figure 1).
Figure 1: Peptide Structure
The structure of a peptoid is different because the replacement group (R) is attached to the nitrogen atoms, rather than the carbons (Figure 2).
Figure 2: Peptoid Structure
Peptoids have an advantage over peptides in that they form stable, helical structures in solution, are highly resistant to degradation, and act as selective antibacterial compounds. They might be used as injectible antibiotics of last resort, as a supplement to conventional antibiotics, as a topical antibiotic ointment or oral rinse, attached to other biomaterials (resin beads) to form antimicrobial surfaces, or as part of a lung surfactant system to treat pneumonia or other lung infections, including those caused from inhaled biological warfare agents. The Barron Lab at Northwestern University has developed a library of peptoids with a wide range of properties. Some are highly antimicrobial; others are extremely
hemolytic – lyse red blood cells. Many of them have properties in between. One of the most promising is Peptoid 5. It has a low MIC (minimum inhibitory concentration) – the lowest concentration of an antimicrobial that will inhibit growth of a microorganism, is not very hemolytic, and only kills mammalian cells in high concentrations (Figure 3a).
E. coli MIC (mM) 3.5 B. subtilis MIC (mM) 0.9
10 Concentration (micromolar) 8 6 4 2 0
Compound 5 Toxicity MTS Assay
LD 10 NIH 3T3 LD 50 NIH 3T3 LD 10 A549 LD 50 A549
Figure 3a: Peptoid 5 Hemolytic Activity
Figure 3b: Peptoid 5 Toxicity
Various assays, such as MTS, have shown that peptoid 5, at concentrations near the MIC, is generally not harmful to mammalian cells such as A549 fibroblasts and H3T3 lung epithelial cells - lethal doses (LD) at which 10 or 50% of the cells will die Figure 3b). Our objective for this research was to determine the specific effects that peptoid 5 has on mammalian cell glycolysis. We worked to refine 96-well plate glucose, lactate, and resazurin enzymatic assays and to determine concentrations, if any, at which the peptoid decreases mammalian cell metabolism, in order to determine its functionality as a potential therapeutic agent. Results are still forthcoming.