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 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

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

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

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

Endothelial Cell in New Blood Vessel

αVβ3 Integrin

RGD-4C

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

Add FITC molecules Add RGD-4C targeting

peptide Analyzing product to

know:– How many FITC?– RGD-4C successfully

added?– How many RGD-4C?

Gen 5 PAMAM Dendrimer

FITC

RG

D-4

C

RGD-4C

RGD-4C

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

Gen 5 PAMAM Dendrimer

FITC

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

Gen 5 PAMAM Dendrimer

FITC

RG

D-4

C

RGD-4C

RGD-4C

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

Mouse Trial: Results

FITC Fluorescence on Tissue Samples from 7/31/06

0

5000

10000

15000

20000

25000

30000

35000

Lung Blood Tumor Kidney Tissue

Flu

ore

sc

en

ce

Control Experimental

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

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 37091

20004 19938

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

Dr. Christine Kelly – Mentor, Chemical Engineering Department

Kelsey Yee – Graduate Student, Chemical Engineering Department

Dr. Kevin Ahern – HHMI Director HHMI Program URISC Program