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Biofunctionalized Nanoparticles for Therapeutics and Live Cell Diagnostics October 10, 2014 1 Team and Facilities Board of Directors • Chad A. Mirkin, PhD • C. Shad Thaxton, PhD • David R. Walt, PhD Management Team • David A. Giljohann, PhD • David S. Snyder, CFO • Sergei Gryaznov, PhD Research Team • • • • • • • • • • • • • • Headquarters Illinois Science + Technology Park, Skokie, IL 9200 sq ft lab and office space Weston Daniel, PhD, Northwestern Richard Kang, PhD, Northwestern Christopher Mader, PhD, Yale Rao Nallagatla, PhD, Penn State Warefta Hasan, PhD, Northwestern Tiffany Halo, PhD, Yale Aleks Radovic-Moreno, PhD, MIT/Harvard Scott Mix, MS Andrew Schook, MS Shweta Iyer, MS Valerie Hilgenberg, MS Blake Tutterow Merideth Burkhart, MS Aakriti Goel, MS Business Development Staff • Pinal Patel, PhD • Ariana Gammel SNAs: A Different Path Spherical Nucleic Acid Greater than 99% cellular uptake -‐ virtually every cell internalized SNAs 7 µm Science, 2006, 312, p1027, ~1000 cita@ons 3 The SNAs Include a Family of DNA and RNA Functionalized Nanoparticles DNA transfected by Gold Nanoparticle - Do not require the use of toxic transfection reagents - Enter 99% of cell population A Unique Path to Gene Regulation Traditional Path: Positively Charged Carrier, Two Materials Required • Delivery agent and nucleic acids needed for transfection • Specialty nucleic acids required • Issues with delivery associated toxicity and distribution Alternate Path: Negatively Charged, Single-Entity SNA Agents • Single-entity agent • Compatible with unmodified nucleic acids • No significant immune response or toxicity SNA Constructs Enter Cells Effectively Without Toxicity Na2ve DNA Low uptake DNA with Lipid Toxicity SNA High uptake, no toxicity PNAS, 2013, 110, p7625 6 Cell Entry is Effective Across Tissues/ Cell Types Cell Lines • • • • • • • Breast (SKBR3, MDAMB-231, AU-565) Brain (U87, LN229, U118) Bladder (HT-1376, 5637, T24) Colon (LS513) Cervix (HeLa, SiHa) Skin (C166, KB, MCF 10A) Kidney (MDCK) • • • • • • • Blood (Sup T1, Jurkat) Leukemia (K562) Liver (HepG2) Kidney (293T) Ovary (CHO) Fibroblast (NIH3T3) Macrophage (RAW264.7) 7 µm HeLa Cells Primary Cells • 15µm Rat Hippocampal Neurons • • • • Brain (Rat Hippocampus Neurons, Astrocytes, Glial Cells) Bladder Blood (PBMC, T-cells) Pancreas (Human β-Islets) Skin (Human) 1 Hour RAW 264.7 Cells 4 Hour SNA Cellular Uptake Depends on DNA Density mol excess PEG Surface Coverage Oligonucleotides per particle Particles (x105) per cell 13 nm Au NP Cellular Uptake Oligonucleotides per particle Mirkin and coworkers, Nano Lett. 2007. Nuclease Nuclease Fluorescence (normalized) SNA Constructs Are Resistant to Degradation dsRNA Spherical RNA Constructs Time (minutes) • Spherical constructs are 10-100 fold more stable than molecular counterparts • Do not require sequence modifications SNA Constructs Cause Minimal Changes in Cellular Gene Expression DharmaFECT1 SNA Constructs Non-treated Spherical Construct Treated vs. Control Number of Differential Genes DharmaFECT Treated vs. Control Up Down Up Down 7 0 206 221 Spherical Constructs Trigger Minimal Innate Immune Response Relative Abundance of IFN-β 1000 Spherical DNA Constructs Lipoplexed DNA 800 600 400 200 0 0.35 0.53 0.71 DNA Concentration (µM) 4 Hour Treatment of RAW Cells with Immuno-Stimulatory DNA Decreasing Oligonucleotide Density Increases Interferon-β Production Relative Abundance of IFN-β / Internalized DNA 1800 1500 1200 900 600 300 0 12.8 5.9 2.1 Oligonucleo@de Density (pmol/cm2) 4 Hour Treatment of RAW Cells DNA/OEG Co-loaded SNAs In collaboration with Curt Horvath, Northwestern University SNA Constructs Cross Blood-Brain-Barrier and Localize to Tumor Sites Controls -Tumor +Tumor Raw Image Photograph Fluorescence Overlay Bcl2L12 siRNA-Au-NP Treated Murine Survival Curve siRNA-Au-NP-Treated Murine Survival Curve Percent survival 150 Beta-Gal (n = 6; Median Survival = 21.0 days) L12-1(n = 6; Median Survival = 21.5 days) L12-2b (n = 5; Median Survival = 26 days) 100 50 0 0 10 20 Days 30 Topically Applied SNA Constructs Penetrate Human Skin 4 hr 20 hr 8 hr 24 hr Histologic skin sec@ons – SNAs in red, cell nuclei in blue 15 AST-‐005: SNA with proprietary an@-‐TNF an@sense oligonucleo@des Thickness of EGFR SNA Construct-treated Skin is decreased by 40% Aquaphor® Only Aquaphor® Only • SNA construct-treated skin shows reduction in Ki-67-positive cells Nonsense SNA Constructs Nonsense SNA Constructs • No apparent signs of toxicity • No upregulation of cytokines/ inflammatory markers EGFR SNA Constructs EGFR SNA Constructs • Scale bar = 100µm Nano-Flares for mRNA Detection Short Duplexes Longer Duplex Formed Flares are Dark Target (mRNA) Flare Released Recognition Sequence: Reporter Sequence: CTT GAG AAA GGG CTG CCA AAA AA-SCCC GAC GGT T-Cy5-5’ Target Region: GAA CTC TTT CCC GAC GGT-5’ Nano-Flares are Taken Up Efficiently and Yield Specific Signal in Response to Genetic Targets 20 µm Survivin Flare Non-Complementary Flare NanoFlare™ is Subject of a Multimillion Dollar Partnership with EMD Millipore ® NanoFlare™: • Revolutionary platform for genetic detection • Works inside living cells • Major advantages for life science researchers EMD Millipore: • • • Major life sciences reagents supplier Multinational sales force focused on bioreagents Compatible instruments and complementary reagents TWO MBP STUDENTS CONTRIBUTED TO THE SUCCESS OF THE PROJECT! Recent Publicity: "If we want to make and sell the best products, we have to invest in the best ideas, just like you do at Northwestern. Your Nanotechnology Ins@tute doesn’t just conduct groundbreaking research; that research has spun off 20 startups and more than 1,800 products, and that means jobs." -‐ President Barack Obama, October 2014 QUESTIONS? [email protected]
