1. Gel Diffusion Experiment STEM ED/CHM Nanotechnology 2012 Presented by Jennifer Welborn

2. Learning Goals In this activity, nanotech participants will: See how food dyes and gelatin are used to model the delivery of nanoscale medicines to cells in the human body See how food dyes and gelatin are used to model the delivery of nanoscale medicines to cells in the human body Measure diffusion distances of 3 different colors of food dye by: Eye, photo image on a computer, ADI software (Analyzing Digital Images) Measure diffusion distances of 3 different colors of food dye by: Eye, photo image on a computer, ADI software (Analyzing Digital Images)

3. Diffusion and Teaching Standards This lab has content which is applicable to various disciplines/standards Physical Science/Chemistry: particle motion theory Biology: passive transport; cellular structure, etc. Ecology/Environmental Science: environmental effects on living systems Math: rates; proportions, data collection, measurement, precision/accuracy

4. Diffusion Diffusion– movement of a substance from a region of higher concentration to a region of lower concentration. Diffusion continues until equilibrium--- the concentration of a substance is equal throughout a space

5. Diffusion and Cells Dissolved particles that are small or non-polar can diffuse through the cell membranes. The process of diffusion is one of the ways in which substances like oxygen, carbon dioxide and water move into and out of cells. Carbon dioxide from the environment diffuses into plant cells

6. Background For Lab Activity The delivery of nanoscale medicines to cells in the human body requires diffusion through tissues, organs and cell membranes The delivery of nanoscale medicines to cells in the human body requires diffusion through tissues, organs and cell membranes This activity will explore the affect of particle size on diffusion rates This activity will explore the affect of particle size on diffusion rates Understanding molecular diffusion through human tissues is important for designing effective drug delivery systems Understanding molecular diffusion through human tissues is important for designing effective drug delivery systems

7. Background Continued Measuring the diffusion of dyes in gelatin is a model for the transport of drugs in the extra-vascular space Measuring the diffusion of dyes in gelatin is a model for the transport of drugs in the extra-vascular space Gelatin is a biological polymeric material with similar properties to the connective extracellular matrix in tumor tissue Gelatin is a biological polymeric material with similar properties to the connective extracellular matrix in tumor tissue Dyes are similar in molecular weight and transport properties to chemotherapeutics Dyes are similar in molecular weight and transport properties to chemotherapeutics

8. Experiment Overview The diffusion of the dyes will be compared to model the effect of molecular weight on movement of molecules in tumors The diffusion of the dyes will be compared to model the effect of molecular weight on movement of molecules in tumors Gelatin will be cut into cylindrical disks, placed in petri dishes and colored solutions will be added to the outer ring Gelatin will be cut into cylindrical disks, placed in petri dishes and colored solutions will be added to the outer ring The distance that the dye particles penetrate into the gelatin disks will be measured over time The distance that the dye particles penetrate into the gelatin disks will be measured over time

9. Lab Prep Collect materials Collect materials –Petri Dishes –Food Dye –Syringes/10 ml graduated cylinders –Paper Cups –Plain Gelatin –Crisco/Petroleum Jelly –Baking Pan –Biscuit cutter Prepare Gel Disks Prepare Gel Disks –Determine amount of water needed to fill up a pan to a depth of 1 cm. –Dissolve gel into cold water (2Pks/Cup/200 ml) –Microwave for 90 Sec. –Pour into pan which has been coated with petroleum jelly and let set.

10. Lab Procedure Gel Disks Gel Disks –Cut disks in pan w/metal bisquit cutter –Remove excess –Move gel disk from pan to petri dish –Top side down! –Centered as best can Adding Dye Adding Dye –Mix dyes in cups –Inject one color/petri dish –No dye on top of gel –No seepage under gel –Do not move dishes after dye inserted –Note volume of dye added

11. Important Details For Procedure Make the red and yellow solutions fairly concentrated. Blue should not be concentrated- it should be strong, but translucent. Make the red and yellow solutions fairly concentrated. Blue should not be concentrated- it should be strong, but translucent. Inject dye towards the outside of the petri dish, not towards the gel. Inject dye towards the outside of the petri dish, not towards the gel. Use an even number of milliliters for the volume of dye. Use an even number of milliliters for the volume of dye. The initial level of dye should not exceed ¾ of the way (7 mm) up the gel. The initial level of dye should not exceed ¾ of the way (7 mm) up the gel. Photograph the gel: same time, same distance, same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax. Photograph the gel: same time, same distance, same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax.

12. Data Collection Method 1-- By eye: measure (in mm) the distance each dye has diffused for each time interval. Record data in a data table or use excel spreadsheet Method 2--Using a digital camera: take photos of each petri dish at the same time each day, 8:45 and 4:45, from the same height and angle

13. Data Collection 3 Food Dyes Start 4 hours Diffusion is first visible

14. Gel Diffusion Analysis

15. Gel Diffusion Analysis Method 1: Determining Rate of Diffusion by Eye Gel Diffusion Analysis Method 1: Determining Rate of Diffusion by Eye –Use graph paper to plot distance (mm) vs time (hours) for each color of dye –The rate is the slope of the line. During the relatively short diffusion time (as in this lab), the relationship between distance and time is somewhat linear. A line of best fit may not have a y-intercept of 0 due to error.

16. Sample Graph of Diffusion Measurements Made by Eye

17. Diffusion Analysis Method 2: Using a Digital Camera Group Pictures by Color in date/time order Group Pictures by Color in date/time order 7-9-16007-10-08007-10-16007-11-0800

18. Pick one color to start Pick one color to start Load the first morning shot Load the first morning shot –Windows Photo Gallery or other image program

19. Using the magnifier, expand the photo Using the magnifier, expand the photo Using a mm ruler, measure from the edge of the gel disk to the inner most edge of the diffusion for each color. Using a mm ruler, measure from the edge of the gel disk to the inner most edge of the diffusion for each color.

20. Calculate the diffusion distances for each dye and for each time period: Calculate the diffusion distances for each dye and for each time period: --Gel diameter measurement (mm) on the computer screen/65 mm = multiplier. --Gel diffusion distance (mm) on screen x multiplier = actual distance. Record calculated diffusion distances for each color and time period in a data table or spread sheet. Record calculated diffusion distances for each color and time period in a data table or spread sheet.

21. Sample Spread Sheet Entry

22. When finished, your table might look something like this When finished, your table might look something like this

23. Create a graph by hand or in excel Create a graph by hand or in excel

24. Calculate Mean Percentage of Diffusion For the last time period measured and for each color of dye, calculate and record the mean percentage of diffusion Use: total distance traveled by dye in mm / 32.5 x 100 = ________% Record the mean percentage of diffusion for each color in your data table or spread sheet

25. Diffusion Analysis Method 3: Using ADI (Analyzing Digital Images Software Download DEW software from: http://umassk12.net/adi/ Click on Analyzing Digital Images

27. Open a picture, then trim the photo to increase processing time

28. Click on the drop down menu

29. Choose Full Image at Selected Resolution Then click on trim and use image

30. Choose this option

31. Draw a line across the diagonal of the petri dish Record petri dish diameter and units Then, click done

32. Select line tool option

33. Zoom in to see diffusion line and edge of gel more clearly Note length of line Draw a line from the edge of the gel to where the diffusion of dye molecules appears to end Click on the blue and red adjustment tools to help you place the blue and red dots at The beginning and end of the line

34. QUALITATIVE OBSERVATION OF DIFFUSION You can also use ADI software to see a qualitative graph of the diffusion of the yellow dye molecules at a particular time. You can compare the qualitative graph with the quantitative measurements. A qualitative graph also helps to see that diffusion is a dynamic process with a trend in movement but no clear end point.

35. Draw a line across the Gel going through the diagonal Choose line tool option

36. Choose graph colors option

37. This graph shows the intensities of red, green and blue pixels along the line drawn across the gel. Notice that around 20/100 the lines level off, indicating edge of diffusion

38. If you turn off all colors but green, you can more easily see that around both 20 and 80 is where the diffusion of the dye molecules tapers off. So, diffusion of the yellow dye particles at this time interval is about 20/100, or.20. Compare this with 1.09 (diffusion distance)/6.03 (gel diameter) =.18

39. Questions to consider Which dyes diffused the fastest? Which dyes diffused the fastest? Does fast diffusion mean greater or poorer retention? Does fast diffusion mean greater or poorer retention? How could diffusion and retention be optimized? This is an important consideration for the delivery of nanoscale medication How could diffusion and retention be optimized? This is an important consideration for the delivery of nanoscale medication