Making and Characterizing
PAMAM Dendrimer Conjugates to
Target Cancer
Erin Rieke
Mentor: Dr. Christine Kelly
Chemical Engineering
Department
Cancer: What is it and why is it hard to
treat?
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Uncontrolled division of cells that forms tumors
Can get into blood system and spread
Cells are not foreign like with infection, etc.
Current treatments – radiotherapy,
chemotherapy, immunotherapy – expose
normal tissue too
Need to target cancer cells
Hard because fundamentally cells are like all
others
Our Strategy: Nanoparticle Based Immunotherapy
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Immunotherapy uses body’s own immune
system to combat cancer
IL-12 used to activate natural killer cell
activity
IL-12 is toxic when given systemically
Possible treatment - nanoparticles
functionalized with IL-12 and targeting agent
Our Strategy: Targeting Angiogenesis
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Tumor growth needs
nutrients
Tumors cause body to grow
new blood vessels –
angiogenesis
New blood vessels branch
from old ones
Vessels lined with
endothelial cells
These endothelial cells
express special markers
Our Strategy: Targeting Angiogenesis
RGD-4C
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αVβ3 Integrin
Endothelial Cell in New Blood
Vessel
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Endothelial cells of new blood
vessels express integrin αVβ3
Tripeptide sequence, arginineglycine-aspartic acid (RGD),
binds to integrin
Many small peptides available
with RGD sequence
RGD-4C, RGD sequence
stablized with two disulfide
bridges, shown to strongest
affinity for integrin αVβ3
PAMAM Dendrimer: Tying it all
Together
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Polyamidoamine (PAMAM)
dendrimer nanoparticle
Ethylenediamine-core
Tertiary amine nitrogens carry
two branched amidoamine
groups
“Dense star” created by
repeated series of reactions
Each reaction adds 2 binding
sites to each tertiary amine
Use “generation 5” dendrimer 5.4 nm with 128 terminal
functional amine groups
My Work: Making the Functionalized
Dendrimer
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How many FITC?
RGD-4C successfully
added?
How many RGD-4C?
RGD-4C
FITC
Gen 5 PAMAM
Dendrimer
RG
D4C
C
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Add FITC molecules
Add RGD-4C targeting
peptide
Analyzing product to
know:
RG
D-4
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Adding FITC to Dendrimer
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Fluorescein isothiocyanate
added to dendrimer and
allowed to react for 18 hrs.
End result is FITC-PAMAM
conjugated dendrimer
Afterwards, sample run on
MALDI-TOF to determine
new molecular mass
Number of FITC on each
dendrimer is determined
Got about 8-10 FITC/
dendrimer
FITC
Gen 5 PAMAM
Dendrimer
RGD Addition and Quantification
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RGD-4C
FITC
Gen 5 PAMAM
Dendrimer
RG
D4C
C
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Once FITC added, need to
add RGD targeting molecule
RGD-4C reacts with amine
termini of dendrimer
Did not use MALDI-TOF to
determine change in molecular
mass
Assumed RGD-4C
successfully added and about
1-2 RGD-4C/ dendrimer
RG
D-4
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Mouse Trials: First Attempt
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Dr. John Mata, co-worker at Vet Med had mice to
be used in cancer treatment experiments
Only had one control mouse and one
experimental mouse
Used dendrimer with about 8-10 FITC/dendrimer
and 1-2? RDG-4C/dendrimer.
Injected 50 uL of dendrimer solution into tail vein
Sacrificed 4 hours later and took samples of
kidney, liver, blood, lung, tumor, and spleen
Mouse Trial: Results
FITC Fluorescence on Tissue Samples from 7/31/06
35000
Fluorescence
30000
25000
20000
Control
Experimental
Tumor
Kidney
15000
10000
5000
0
Lung
Blood
Tissue
Analysis of Results
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All tissue samples fluoresced very small amounts
Not enough FITC/dendrimer and not enough
dendrimer in injection
Fluorescence in experimental mouse congregated in
blood
Maybe no RGD-4C added (more on this later)
Need to optimize dendrimer conjugation before
doing further mice trials
New RGD Peptide sequence
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Decided to use different RGD
peptide, cyclic RGD
New RGD attaches to carboxylic
acid groups, not amines
Converted all amines to -COOH
groups after adding FITC then add
cyclic-RGD
Attempted once, but MALDI-TOF
showed no cyclic-RGD appeared to
attach
Maybe no RGD-4C attached in the
first experiment (did not explicitly
test before injecting
MALDI-TOF Results
38875
20004
37091
19938
Next Steps
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Successfully add RGD peptide
Use cell cultures to perform positive and
negative control experiments for targeting
Attach IL-12 and begin mouse model
experiments to determine effectiveness
Thank You
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Dr. Christine Kelly – Mentor, Chemical
Engineering Department
Kelsey Yee – Graduate Student, Chemical
Engineering Department
Dr. Kevin Ahern – HHMI Director
HHMI Program
URISC Program