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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?. Uncontrolled division of cells that forms tumors Can get into blood system and spread

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making and characterizing pamam dendrimer conjugates to target cancer

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
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
our strategy nanoparticle based immunotherapy
Our Strategy: Nanoparticle Based Immunotherapy
  • 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
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
our strategy targeting angiogenesis5
Our Strategy: Targeting Angiogenesis
  • Endothelial cells of new blood vessels express integrin αVβ3
  • 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 αVβ3
pamam dendrimer tying it all together
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
my work making the functionalized dendrimer
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?
adding fitc to dendrimer
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
rgd addition and quantification
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
mouse trials first attempt
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
analysis of results
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
new rgd peptide sequence
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
maldi tof results
MALDI-TOF Results

38875

37091

20004

19938

next steps
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
thank you
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