Making and Characterizing PAMAM Dendrimer Conjugates to Target Cancer

1
Making and Characterizing PAMAM Dendrimer
Conjugates to Target Cancer

• Erin Rieke
• Mentor Dr. Christine Kelly
• Chemical Engineering Department

2
Cancer What is it and why is it hard to treat?

• 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

3
Our Strategy Nanoparticle Based Immunotherapy

• Immunotherapy uses bodys 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

4
Our Strategy Targeting Angiogenesis

• 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

5
Our Strategy Targeting Angiogenesis

• Endothelial cells of new blood vessels express
  integrin aVß3
• Tripeptide sequence, arginine-glycine-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
  aVß3

6
PAMAM Dendrimer Tying it all Together

• 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

7
My Work Making the Functionalized Dendrimer

• Add FITC molecules
• Add RGD-4C targeting peptide
• Analyzing product to know
• How many FITC?
• RGD-4C successfully added?
• How many RGD-4C?

8
Adding FITC to Dendrimer

• 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

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Percent Acetylation

• React dendrimer, in methanol, with acetic
  anhydride
• Have excess triethylamine to neutralize acid
  created in reaction
• Take NMR and compare to NMRs of known
  actetylations
• Trying for 70 acetylation

10
Determining FITC Attachment

• Standard curve made by measuring fluorescence of
  known concentrations
• Product fluorescence was read
• Very odd results obtained 0.1 FITC/Dendrimer
• Why?

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Determining FITC Attachment

• FITC fluorescence is pH dependent
• Surface of dendrimer is covered with amines,
  creating basic pH
• pH of standards was about 7.4, not basic
• Solution Use different fluorescent molecule
• TAMARA does not have a pH dependence and
  fluoresces red

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RGD Addition and Quantification

• 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

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RGD-4C Addition and Quantification

• Used gel permeation chromatography (GPC) to
  determine final composition
• Ran product through after every step to determine
  molecular weight
• Backed-out composition knowing how much dendrimer
  we started with

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Mouse Trials First Attempt

• 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

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Mouse Trial Results
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Analysis of Results

• 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

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New RGD Peptide sequence

• 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

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MALDI-TOF Results
38875
37091
20004
19938
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Next Steps

• 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

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Thank You

• Dr. Christine Kelly Mentor, Chemical
  Engineering Department
• Kelsey Yee Graduate Student, Chemical
  Engineering Department
• Dr. Kevin Ahern HHMI Director
• HHMI Program
• URISC Program

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Does the Targeting Work?

• Needed to test targeting
• Grew two different cell lines
• SAOS, dog osteosarcoma line
• Do not express integrin
• SB-HAS, endothelial cell line
• Express integrin
• Exposed cells to dendrimer
• Analyzed with fluorescent microscopy

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Results First Trial

• Integrin positive cells, SB-HAS, show
  fluorescence all throughout the cells
• Integrin positive cells show fluorescence
  bordering the cells
• Results bode well for treatment method

SB-HSA cells exposed to dendrimer solution for 30
min and washed once with PBS
SAOS cells exposed to dendrimer solution for 30
min and washed once with PBS