Gel Diffusion Gel Diffusion ExperimentExperiment

STEM ED/CHMSTEM ED/CHM

Nanotechnology 2014Nanotechnology 2014

Presented by Jennifer Welborn Presented by Jennifer Welborn

Learning GoalsLearning Goals

In this activity, nanotech participants will:In this activity, nanotech participants will: See how food dyes and gelatin are See how food dyes and gelatin are

used to model the delivery of nanoscale used to model the delivery of nanoscale medicines to cells in the human bodymedicines to cells in the human body

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

Diffusion and Teaching StandardsThis 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 ActivityBackground For Lab Activity

The delivery of nanoscale medicines The delivery of nanoscale medicines to cells in the human body requires to cells in the human body requires diffusion through tissues, organs and diffusion through tissues, organs and cell membranescell membranes

This activity will explore the affect of This activity will explore the affect of particle size on diffusion ratesparticle size on diffusion rates

Understanding molecular diffusion Understanding molecular diffusion through human tissues is important through human tissues is important for designing effective drug delivery for designing effective drug delivery systems systems

Background ContinuedBackground Continued

Measuring the diffusion of dyes in Measuring the diffusion of dyes in gelatin is a model for the transport of gelatin is a model for the transport of drugs in the extra-vascular space drugs in the extra-vascular space

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

Dyes are similar in molecular weight Dyes are similar in molecular weight and transport properties to and transport properties to chemotherapeuticschemotherapeutics

Experiment OverviewExperiment Overview

Gelatin will be cut into cylindrical disks, Gelatin will be cut into cylindrical disks, placed in Petri dishes and colored placed in Petri dishes and colored solutions will be added to the outer ringsolutions will be added to the outer ring

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

The diffusion of the dyes will be The diffusion of the dyes will be compared to model the effect of compared to model the effect of molecular weight on movement of molecular weight on movement of molecules in tumors molecules in tumors

Lab PrepLab Prep

Collect materialsCollect materials– Petri DishesPetri Dishes– Food DyeFood Dye– Syringes/10 ml Syringes/10 ml

graduated cylindersgraduated cylinders– Paper CupsPaper Cups– Plain GelatinPlain Gelatin– Crisco/Petroleum JellyCrisco/Petroleum Jelly– Baking PanBaking Pan– Biscuit cutterBiscuit cutter

Prepare Gel DisksPrepare Gel Disks– Determine amount of Determine amount of

water needed to fill up a water needed to fill up a pan to a depth of 1 cm. pan to a depth of 1 cm.

– Dissolve gel into cold Dissolve gel into cold water (2Pks/Cup/200 ml)water (2Pks/Cup/200 ml)

– Microwave for 90 Sec.Microwave for 90 Sec.– Pour into pan which has Pour into pan which has

been coated with been coated with petroleum jelly and let petroleum jelly and let set.set.

Lab ProcedureLab Procedure

Gel DisksGel Disks– Cut disks--bisquit Cut disks--bisquit

cuttercutter– Thin coating of Thin coating of

Petroleum jelly on Petroleum jelly on inside bottom of Petri inside bottom of Petri dishdish

– Put gel disk –top side Put gel disk –top side down and centered- down and centered- on bottom of dishon bottom of dish

– Gently press disk to Gently press disk to secure secure

Adding DyeAdding Dye– Mix dyes in cupsMix dyes in cups– Inject one Inject one

color/petri dishcolor/petri dish– No dye on top of gelNo dye on top of gel– No seepage under No seepage under

gelgel– Do not move dishes Do not move dishes

after dye insertedafter dye inserted

Important Details For ProcedureImportant 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, Inject dye towards the outside of the petri dish,

not towards the gel.not towards the gel. Photograph the gel: same time, same distance, Photograph the gel: same time, same distance,

same ambient lighting, flash off, cover off, same same ambient lighting, flash off, cover off, same sequence. Keep camera parallel to gel (do not tilt) sequence. Keep camera parallel to gel (do not tilt) to avoid parallax. to avoid parallax.

Data CollectionData Collection

Method 1-- By eyeMethod 1-- By eye: measure (in mm) : measure (in mm) the distance each dye has diffused for the distance each dye has diffused for each time interval. Record data in a each time interval. Record data in a data table or use excel spreadsheet data table or use excel spreadsheet

Method 2--Using a digital cameraMethod 2--Using a digital camera: take : take photos of each petri dish at the same photos of each petri dish at the same time each day, 8:45 and 4:45, from the time each day, 8:45 and 4:45, from the same height and angle same height and angle

Applications Applications

Nano-medicine: Targeted Nano-medicine: Targeted TherapiesTherapies

Nanoparticles diffuse into cancer cells then heated in a magnetic field Nanoparticles diffuse into cancer cells then heated in a magnetic field to weaken the cells. Chemotherapy is more effective on the to weaken the cells. Chemotherapy is more effective on the weakened cells. weakened cells.

The dye in blue jeans or ballpoint pens has also been paired with The dye in blue jeans or ballpoint pens has also been paired with gold nanoparticles to fight cancer. This dye, known as gold nanoparticles to fight cancer. This dye, known as phthalocyaninephthalocyanine, reacts with light. The nanoparticles take the dye , reacts with light. The nanoparticles take the dye directly to cancer cells while normal cells reject the dye. Once the directly to cancer cells while normal cells reject the dye. Once the particles are inside, scientists "activate" them with light to destroy particles are inside, scientists "activate" them with light to destroy the cancer. the cancer.

Similar therapies have existed to treat skin cancers with light-Similar therapies have existed to treat skin cancers with light-activated dye, but scientists are now working to use nanoparticles activated dye, but scientists are now working to use nanoparticles and dye to treat tumors deep in the body.and dye to treat tumors deep in the body.

http://science.howstuffworks.com/life/human-biology/gold

nanotech1.htmnanotech1.htm

Nanomedicine, continuedNanomedicine, continued

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

http://umassk12.net/nano/

Targeted Delivery to Targeted Delivery to TumorsTumors

• 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-Nanomedicine connection-youtubeyoutube

Youtube video made by the Center Youtube video made by the Center for Hierarchical Manufactoring at for Hierarchical Manufactoring at UMASS, Amherst:UMASS, Amherst:

http://www.youtube.com/watch?v=bUvi5eQhPTc

5:40-7:40 shows specific uses of 5:40-7:40 shows specific uses of diffusion of nano-scale particles in diffusion of nano-scale particles in medicine. The rest of the video is medicine. The rest of the video is AWESOME!AWESOME!

Gel Diffusion Lab- ApplicationsGel 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 AnalysisGel Diffusion Analysis

Method 1: Using ADI (Analyzing Digital Images) Software Download DEW software from: http://umassk12.net/adi/

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, clickdone

Select line tool option

Zoom in to see diffusion lineand edge of gel more clearly

Note length of line

Draw a line from the edgeof 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 atThe 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 theGel 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 drawnacross 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 tapersoff. 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 Analysis/Discussion 1.1. What do the data tell us about diffusion?What do the data tell us about diffusion?

2.2. Does the addition of two dyes at the same time affect the Does the addition of two dyes at the same time affect the rate of each?rate of each?

3. What implications does this have for the delivery of 3. What implications does this have for the delivery of medicine into cells? medicine into cells?

Analysis Method 2: Determining Analysis Method 2: Determining Diffusion Rate by Eye Diffusion Rate by Eye

– Use graph paper or a graphing program Use graph paper or a graphing program to plot distance (mm) vs time (hours) for to plot distance (mm) vs time (hours) for each color of dyeeach color of dye

– The rate is the slope of the line. At the The rate is the slope of the line. At the beginning of the lab, the relationship beginning of the lab, the relationship between distance and time is somewhat between distance and time is somewhat linear. Over time, the rate decreases linear. Over time, the rate decreases and the line levels off. and the line levels off.

Analysis Method 3: Using a Digital Analysis Method 3: Using a Digital CameraCamera

Group Pictures by Color in date/time Group Pictures by Color in date/time

orderorder

7-9-1600 7-10-0800 7-10-1600 7-11-0800

Pick one color to startPick one color to start Load the first morning shotLoad the first morning shot

– Windows Photo Gallery or other image Windows Photo Gallery or other image programprogram

Using the magnifier, expand the Using the magnifier, expand the photophoto

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

Calculate the diffusion distances for Calculate the diffusion distances for each dye and for each time period: each dye and for each time period:

--Gel diameter measurement (mm) on the --Gel diameter measurement (mm) on the computer screen/65 mm = multiplier. computer screen/65 mm = multiplier.

--Gel diffusion distance (mm) on screen x --Gel diffusion distance (mm) on screen x multiplier = actual distance.multiplier = actual distance.

Record calculated diffusion distances Record calculated diffusion distances for each color and time period in a for each color and time period in a data table or spread sheet. data table or spread sheet.

Sample Spread Sheet Entry

When finished, your table might When finished, your table might look something like thislook 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 colorof 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 Additional Questions to ConsiderConsider

Which dyes diffused the fastest? Which dyes diffused the fastest? Does fast diffusion mean greater or Does fast diffusion mean greater or

poorer retention?poorer retention? How could diffusion and retention be How could diffusion and retention be

optimized? This is an important optimized? This is an important consideration for the delivery of consideration for the delivery of nanoscale medication nanoscale medication

Molecular Weights of the Molecular Weights of the DyesDyes

Red #40 molar mass- 879.86 g/molRed #40 molar mass- 879.86 g/mol Yellow #5: molar mass- 453.27 Yellow #5: molar mass- 453.27

g/molg/mol Blue #1: molar mass--792.84 g/molBlue #1: molar mass--792.84 g/mol