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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?. 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

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  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 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

  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 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

  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

  9. 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

  10. 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

  11. Mouse Trial: Results

  12. 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

  13. 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

  14. MALDI-TOF Results 38875 37091 20004 19938

  15. 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

  16. 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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