Gel Diffusion Experiment STEM ED/CHM Nanotechnology 2014 Presented by Jennifer Welborn

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  Measure diffusion distances of 3 different colors of food dye by: Eye, photo image on a computer, ADI software (Analyzing Digital Images)

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

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

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

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

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: biological polymeric material with similar properties to the connective extracellular matrix in tumor tissue Gelatin: 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

Experiment Overview 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 diffuse into the gelatin disks will be measured over time The distance that the dye particles diffuse into the gelatin disks will be measured over time 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

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.

Lab Procedure Gel Disks Gel Disks –Cut disks--bisquit cutter –Thin coating of Petroleum jelly on inside bottom of Petri dish –Put gel disk –top side down and centered- on bottom of dish –Gently press disk to secure 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

Important Details For Procedure Make the dye solutions according to directions. Make the dye solutions according to directions. 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. Photograph the gel: same time, same distance, same ambient lighting, flash off, cover off, same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax. Photograph the gel: same time, same distance, same ambient lighting, flash off, cover off, same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax.

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

Applications

Nano-medicine: Targeted Therapies Nanoparticles diffuse into cancer cells then heated in a magnetic field to weaken the cells. Chemotherapy is more effective on the weakened cells. Nanoparticles diffuse into cancer cells then heated in a magnetic field to weaken the cells. Chemotherapy is more effective on the weakened cells. The dye in blue jeans or ballpoint pens has also been paired with gold nanoparticles to fight cancer. This dye, known as phthalocyanine, reacts with light. The nanoparticles take the dye directly to cancer cells while normal cells reject the dye. Once the particles are inside, scientists "activate" them with light to destroy the cancer. The dye in blue jeans or ballpoint pens has also been paired with gold nanoparticles to fight cancer. This dye, known as phthalocyanine, reacts with light. The nanoparticles take the dye directly to cancer cells while normal cells reject the dye. Once the particles are inside, scientists "activate" them with light to destroy the cancer. Similar therapies have existed to treat skin cancers with light-activated dye, but scientists are now working to use nanoparticles and dye to treat tumors deep in the body. Similar therapies have existed to treat skin cancers with light-activated dye, but scientists are now working to use nanoparticles and dye to treat tumors deep in the body. nanotech1.htm nanotech1.htm

Nanomedicine, continued The next 3 slides are from Professor Jonathan Rothstein’s presentation. The full presentation can be found at: The next 3 slides are from Professor Jonathan Rothstein’s presentation. The full presentation can be found at:

Targeted Delivery to Tumors Goal is to inject treatment far from tumor and have large accumulation in tumor and minimal accumulation in normal cells/organs.

Example of Current Cancer Treatment Tumor penetration is a key issue for successful chemotherapy

Nanoparticle use in Cancer Treatments Because of their small size, nanoparticles can pass through interstitial spaces between necrotic and quiescent cells. Tumor cells typically have larger interstitial spaces than healthy cells Particles collect in center bringing therapeutics to kill the tumor from inside out.

Nanomedicine connection- youtube Youtube video made by the Center for Hierarchical Manufactoring at UMASS, Amherst: Youtube video made by the Center for Hierarchical Manufactoring at UMASS, Amherst: QhPTc QhPTc QhPTc QhPTc 5:40-7:40 shows specific uses of diffusion of nano-scale particles in medicine. The rest of the video is AWESOME! 5:40-7:40 shows specific uses of diffusion of nano-scale particles in medicine. The rest of the video is AWESOME!

Gel Diffusion Lab- Applications Turn and Talk How might you use this lab or parts of it in your classroom? What challenges might you have for implementation? How might you address those challenges?

Gel Diffusion Analysis

Method 1: Using ADI (Analyzing Digital Images) Software  Download DEW software from:  Click on Analyzing Digital Images

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

Click on the drop down menu

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

Choose this option

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

Select line tool option

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

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.

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

Choose graph colors option

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

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

Analysis/Discussion 1. What do the data tell us about diffusion? 2. Does the addition of two dyes at the same time affect the rate of each? 3. What implications does this have for the delivery of medicine into cells?

Analysis Method 2: Determining Diffusion Rate by Eye Analysis Method 2: Determining Diffusion Rate by Eye –Use graph paper or a graphing program to plot distance (mm) vs time (hours) for each color of dye –The rate is the slope of the line. At the beginning of the lab, the relationship between distance and time is somewhat linear. Over time, the rate decreases and the line levels off.

Analysis Method 3: Using a Digital Camera Group Pictures by Color in date/time order Group Pictures by Color in date/time order

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

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.

 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.

Sample Spread Sheet Entry

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

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

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

Additional 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

Molecular Weights of the Dyes Red #40 molar mass g/mol Red #40 molar mass g/mol Yellow #5: molar mass g/mol Yellow #5: molar mass g/mol Blue #1: molar mass g/mol Blue #1: molar mass g/mol