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 α V β 3 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 α V β 3

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 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?

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

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

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?

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

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

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

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

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

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

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

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