Transcript Gelatin Diffusion Experiment

Gelatin Diffusion Experiment
Nanotechnology in Medicine
Neil S. Forbes
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
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
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
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.
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?
Start
3 hours
Diffusion is
first visible
8 hours
Green Food Color
12 hours
48 hours
24 hours
60 hours
36 hours
72 hours
Final
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.
Time
8
24
48
72
Relative Dye
Penetration (mm) Gel Width (mm)
11
146
19
140
25
123
44
178
Image Analysis II
• Calculate the absolute penetration depth
• We know that the gel is 60mm wide
• The penetration distance is:
60mm
Penetration Depth = Relative Penetration
Gel Width
Time
8
24
48
72
Relative Dye
Absolute
Penetration (mm) Gel Width (mm) Dye Penetration (mm)
11
146
4.5
19
140
8.1
25
123
12.2
44
178
14.8
Image Analysis III
• Plot the distance vs. time
16.0
Distance (mm)
12.0
8.0
4.0
0.0
0
20
40
Time (hours)
60
80
Image Analysis IV
• The linear diffusion rate is the slope of the line:
• 0.16 mm/hr or 3.8 mm/day
20.0
Distance (mm)
16.0
12.0
8.0
y = 0.1601x + 3.8379
4.0
0.0
0
20
40
Time (hours)
60
80
Try image analysis yourself!
Dye Composition
• Yellow: Tartrazine
• Red:
Allura Red
• Blue: Brilliant Blue
Allura Red
Erthrosine
Allura Red
Tartrazine
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
Start
1
3 Hours
8 Hours
0.8
24 Hours
Concentration
36 Hours
48 Hours
0.6
60 Hours
72 Hours
0.4
0.2
0
0
0.2
0.4
0.6
Distance
0.8
1
Questions to Consider
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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?
Relation to Therapy
Results
• Diffusion is very slow (millimeters per
hour)
• The physical properties of a dye (or drug)
affect the diffusion rate
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