Gelatin Diffusion Experiment

1. Gelatin Diffusion Experiment Nanotechnology in Medicine Neil S. Forbes

2. Background • The delivery of nanoscale medicines to cells in the human body requires diffusion through tissues, organs and cell membranes • This activity explores the affect of different diffusion rates • Understanding molecular diffusion through human tissues is important for designing effective drug delivery systems

3. Introduction • Measuring the diffusion of dyes in gelatin illustrates 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 • Dyes are similar in molecular weight and transport properties to chemotherapeutics • Their concentration can be easily determined simply by color intensity • Green food dye contains tartrazine (FD&C yellow #5) and brilliant blue FCF (FD&C blue #1), which have molecular formulae of C16H9N4Na3O9S2 and C37H34N2Na2O9S3, and absorb yellow light at 427nm and blue light at 630nm

4. Experiment Overview • The diffusion of the dyes is measured to demonstrate the effect of molecular weight on transport in tumors • Gelatin will be formed into cylindrical shapes in Petri dishes and colored solutions will be added to the outer ring • Over several days the distance that the dyes and particles penetrate into the gelatin cylinders will be measured

5. Experimental Setup • Gelatin cylinders are formed in Petri dishes. This represents tumor tissue. • Food dye is added to the space surrounding the gelatin. This represents the lumen of blood vessels. • Each day, two images are of dye diffusion are acquired. This captures the penetration of nanoparticles or drugs in to the tumor. • The rate of diffusion is calculated from the images. • The diffusion rates of different dyes can be compared.

6. Questions to consider • What did you expect to happen? • Which dyes do you expect to penetrate better? • Does fast diffusion mean greater or poorer retention? • Why does diffusion matter? • Does retention matter? • Could diffusion and retention be optimized?

7. Start 3 hours Diffusion is first visible 8 hours Green Food Color

8. 12 hours 48 hours 24 hours 60 hours 36 hours 72 hours

9. Final

10. Image Analysis • Display the images on a computer screen. • Distances in the images need to be calibrated. • Use a ruler to measure the depth of penetration and the width of the gel on the screen.

11. Image Analysis II • Calculate the absolute penetration depth • We know that the gel is 60mm wide • The penetration distance is:

12. Image Analysis III • Plot the distance vs. time

13. Image Analysis IV • The linear diffusion rate is the slope of the line: • 0.16 mm/hr or 3.8 mm/day

14. Try image analysis yourself!

15. Dye Composition • Yellow: Tartrazine Allura Red • Red: Allura Red Erthrosine • Blue: Brilliant Blue Allura Red

16. Tartrazine

19. Comparison to Theory • Precise image analysis can quantify the dye concentration as a function of position and time • This analysis fit well with theoretical predictions

20. Questions to Consider • Are the results expected? • Which dyes penetrated better? • Do your results make sense? • Which would penetrate the best? • Which would have the best retention? • Which would be the best drug (based on transport alone)? • Do you have enough information to answer these questions? • What else would you need to know? • How could nanotechnology be used to optimize drug diffusion and retention?

21. Relation to Therapy

22. Results • Diffusion is very slow (millimeters per hour) • The physical properties of a dye (or drug) affect the diffusion rate

23. Implications • Understanding the relation between diffusion and convective delivery (through the vasculature) is essential • The properties of delivery systems should be carefully tailored to enhance drug penetration and retention