Nanotechnology Bio

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Nanotechnology Bio

  1. 1. Biomedical Applications of Nanotechnology
  2. 2. What is Nanotechnology? The creation of useful, functional materials, devices, and systems through: 1. Controlling and manipulating matter on the nanometer-length scale (1-100 nm), and 2. Exploiting novel phenomena and properties (physical, chemical, biological, mechanical, electrical) at the nanoscale. “Going Small for Big Advances” “Going Small for Big Advances”
  3. 3. Potential market for nanotechnology ? $1 trillion business within the next 10 to 15 years.
  4. 4. Nanotechnology in Medicine • Biological imaging for medical diagnostics. • Advanced drug delivery systems. • Biosensors for airborne chemicals or other toxins. • Regenerative medicine: More durable, rejection-resistant artificial tissues and organs. NANOMEDICINE NANOMEDICINE
  5. 5. Targeted Drug Delivery J Leukoc Biol 2005;78:585
  6. 6. Cardiovascular Obesity / Disease Diabetes Risk Factors Inflammation Nanotechnology Tumor Neurological Angiogenesis Disorder / Metastasis
  7. 7. Development of In Vivo and In Vitro Inflammatory Disease Models
  8. 8. In Vivo Brain Inflammation Models • Environmental Toxicants-Mediated Brain Inflammation. • Bacterial Infection-Mediated Brain Inflammation.
  9. 9. Environmental Toxicants & Brain Inflammation Isolation of brain regions C57/BL6 Mouse Determination of pro-inflammatory mediators - Cytokines: TNF-α, IL-1β, IL-6 - Chemokine: MCP-1 Chlorpyrifos - Adhesion molecules: E-selectin, ICAM-1, VCAM-1 [OP Pesticide]
  10. 10. 20 * Control (TNF-α mRNA/GAPDH mRNA) CPF, 8 h CPF, 16 h Relative Fold Induction 15 CPF, 24 h * 10 * 5 * * * * * * * 0 HIP COR STR CER
  11. 11. 25 (TNF-α mRNA/GAPDH mRNA) Control CPF, 20 mg/kg * CPF, 70 mg/kg 20 Relative Fold Induction CPF, 140 mg/kg 15 10 * * * 5 * * * 0 HIP COR STR CER
  12. 12. 6 Control * CPF, 4 h (IL-6 mRNA/GAPDH mRNA) 5 CPF, 8 h Relative Fold Induction CPF, 24 h 4 * * 3 * * * 2 * * 1 0 HIP COR STR CER
  13. 13. 7 Control (IL-6 mRNA/GAPDH mRNA) CPF, 20 mg/kg * 6 CPF, 70 mg/kg Relative Fold Induction CPF, 140 mg/kg 5 4 * * 3 * * 2 1 0 HIP COR STR CER
  14. 14. 6 * Control (MCP-1 mRNA/GAPDH mRNA) CPF, 8 h 5 CPF, 16 h Relative Fold Induction CPF, 24 h 4 * * 3 * * * 2 1 0 HIP STR CER
  15. 15. 5 (MCP-1 mRNA/GAPDH mRNA) Control * CPF, 20 mg/kg * CPF, 70 mg/kg 4 Relative Fold Induction CPF, 140 mg/kg 3 * * 2 1 0 HIP STR CER
  16. 16. 3.0 * (E-selectin mRNA/GAPDH mRNA) Control * CPF, 70 mg/kg 2.5 Relative Fold Induction * 2.0 1.5 1.0 0.5 0.0 HIP COR STR CER
  17. 17. Bacterial Infection & Brain Inflammation Isolation of brain regions C57/BL6 Mouse Determination of pro-inflammatory mediators - Cytokines: TNF-α, IL-1β, IL-6 - Chemokine: MCP-1 Lipopolysaccharide - Adhesion molecules: E-selectin, ICAM-1, VCAM-1 [LPS]
  18. 18. 40 30 * Control (TNF-α mRNA/GAPDH mRNA) 35 LPS * 25 Relative Fold Induction * 30 * * 20 25 * 20 15 15 10 10 5 5 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  19. 19. 35 12 (IL-6 mRNA/GAPDH mRNA) 30 Control * * LPS Relative Fold Induction 25 9 20 * 6 15 * 10 * 3 * 5 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  20. 20. 25 6 * Control * (IL-1β mRNA/GAPDH mRNA) LPS 5 20 Relative Fold Induction 4 * 15 3 * * 10 * 2 * 5 1 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  21. 21. 120 20 Control (MCP-1 mRNA/GAPDH mRNA) LPS * * 100 Relative Fold Induction * 15 80 * * 60 10 40 * * 5 20 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  22. 22. 15 Control 10 (ICAM-1 mRNA/GAPDH mRNA) * LPS * * Relative Fold Induction 12 * 8 9 6 * * 6 * 4 * 3 2 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  23. 23. 20 15 (E-selectin mRNA/GAPDH mRNA) Control LPS * * Relative Fold Induction 12 15 * * 9 * 10 * * 6 5 * 3 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  24. 24. In Vitro Brain Inflammation Models • Brain Microvascular Endothelial Cells (BMEC) • Astrocytes • Microglia
  25. 25. Pro-inflammatory Stimuli-Mediated Inflammation in Brain Cells Pro-inflammatory Stimuli [100 ng/ml of LPS] Culture Media Brain Cells Determination of pro-inflammatory mediators - Cytokines: TNF-α, IL-1β, IL-6 - Chemokine: MCP-1 - Adhesion molecules: E-selectin, ICAM-1
  26. 26. Brain Microvascular Endothelial Cells (bEnd.3) 120 10 * (TNF-α mRNA/GAPDH mRNA) (IL-1β mRNA/GAPDH mRNA) 100 * 8 Relative Fold Induction Relative Fold Induction 80 6 60 * 4 40 * 2 20 * * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  27. 27. Brain Microvascular Endothelial Cells (bEnd.3) 20 100 * (MCP-1 mRNA/GAPDH mRNA) * (IL-6 mRNA/GAPDH mRNA) 80 Relative Fold Induction Relative Fold Induction 15 * 60 * 10 * 40 * 5 20 * * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  28. 28. Brain Microvascular Endothelial Cells (bEnd.3) 30 * 12 (E-selectin mRNA/GAPDH mRNA) (ICAM-1 mRNA/GAPDH mRNA) * 25 Relative Fold Induction Relative Fold Induction * 9 20 * 15 6 * 10 3 5 * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  29. 29. Relative Fold Induction (TNF-α mRNA/GAPDH mRNA) 0 10 20 30 40 50 Control * LPS Relative Fold Induction 0 (IL-1β mRNA/GAPDH mRNA) 1 2 800 900 1000 1100 Microglia (BV-2) Control * LPS
  30. 30. Relative Fold Induction (IL-6 mRNA/GAPDH mRNA) 0 1 2 6500 7000 7500 8000 Control * LPS Relative Fold Induction 0 (MCP-1 mRNA/GAPDH mRNA) 10 20 30 40 50 60 70 80 90 Microglia (BV-2) Control * LPS
  31. 31. Relative Fold Induction (TNF-α mRNA/GAPDH mRNA) 0 1 2 330 360 390 420 Control * LPS Relative Fold Induction (IL-6 mRNA/GAPDH mRNA) 0 5 10 15 20 25 30 35 40 45 Astrocytes (C6) Control * LPS
  32. 32. Relative Fold Induction (MCP-1 mRNA/GAPDH mRNA) 0 10 20 30 40 50 60 70 80 Control * Astrocytes (C6) LPS
  33. 33. In Vitro Ischemia/Reperfusion Model A Novel Design of Double- Layer Parallel-Plate Flow Chamber & Its Biomedical Application
  34. 34. In Vitro Flow Chamber Systems Cone-Plate Orbital Shaker Artificial Capillary Parallel-Plate
  35. 35. Parallel-plate flow chambers (PPFC) have been most commonly used for its simplicity of concept x y l h w z Flow enters the parallel plates at the origin and exits where x equals the length of the chamber, l
  36. 36. Conventional PPFCs have shown weaknesses and problems in several aspects of its design
  37. 37. To eliminate these problems, we designed and developed a new double-layer PPFC • Accepts up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. • Provides a total of 96 cm2 cell monolayer per chamber. • Placing glass slides in series shortens the duration of procedure. • The multilayer design only requires 2D cutting, which is easier and faster to manufacture and modify.
  38. 38. The system becomes much simpler with the new chamber.
  39. 39. The new double-layer PPFC consists of separate layers of different materials and thicknesses Acrylic sheets of 0.08 inch thickness Acrylic sheets of 0.5 inch thickness Each acrylic layer was cut by Laser Computer Aided Modeling and Manufacture (LaserCAMM) machine. The system is a computerized laser cutter that uses a laser beam to cut sheet materials into intricate patterns with a high degree of accuracy.
  40. 40. The new double-layer PPFC consists of separate layers of different materials and thicknesses Silicone gaskets of 0.03 inch thickness Silicone gasket of 0.01 inch thickness Silicone gaskets of 0.03 inch thickness serve as a firm grip for glass slides. The silicone gasket in the middle constitutes the channel height, h, and the width, w.
  41. 41. The new double-layer PPFC consists of separate layers of different materials and thicknesses Glass slides to fill up space Glass slides which will have cells seeded Up to four glass slides can be entered in a chamber. Glass slides or endothelial monolayers are placed between the gasket in the middle. Placing endothelial monolayers on both sides of channel minimizes pressure loss while having a larger effective area.
  42. 42. The new double-layer PPFC consists of separate layers of different materials and thicknesses Media enters through the inlets. Fills up a small reservoir formed in the gasket. Spreads evenly across width through a thin slit. Flows across the endothelial monolayer. Escapes the chamber through the thin slit, the small reservoir, and the outlets.
  43. 43. The new double-layer PPFC consists of separate layers of different materials and thicknesses To set up the chamber bubble-free, the layers are installed in the order from the bottom to the top layers where the flow channel, reservoirs are filled up with media by means of syringe as each layer is piled up.
  44. 44. A flow loop system provides a constant hydrostatic pressure to the PPFC Upper Reservoir Flow Flow Meter Peristaltic Pump Lower PPFC Reservoir
  45. 45. • The streamlines near the lateral walls were not disturbed ensuring that the lateral wall effects are negligible. • The chamber clearly applies a uniform magnitude of shear stress throughout the entire surface where endothelial cell monolayer will be placed.
  46. 46. RBE4 HMEC-1 Static Flow
  47. 47. 1.4 1.2 Static 1.2 Flow (Relative Fold Induction) (Relative Fold Induction) IL-6 Gene Expression Gene Expression 0.9 1.0 0.8 0.6 * * 0.6 * 0.4 0.3 * 0.2 0.0 0.0 Static Flow ICAM-1 VCAM-1 E-selectin
  48. 48. In Vitro Ischemia/Reperfusion Model 7 Normal Flow (IL-6 mRNA/β-Actin mRNA) Relative Fold Induction 6 Ischemia/Reperfusion * 5 4 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  49. 49. In Vitro Ischemia/Reperfusion Model 5 Normal Flow * (MCP-1 mRNA/β-Actin mRNA) Ischemia/Reperfusion 4 * Relative Fold Induction 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  50. 50. In Vitro Ischemia/Reperfusion Model 3 (ICAM-1 mRNA/β-Actin mRNA) Normal Flow Ischemia/Reperfusion * Relative Fold Induction 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  51. 51. In Vitro Ischemia/Reperfusion Model 6 (VCAM-1 mRNA/β-Actin mRNA) Normal Flow Relative Fold Induction 5 Ischemia/Reperfusion * 4 3 * 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  52. 52. In Vitro Ischemia/Reperfusion Model 5 (E-selectin mRNA/β-Actin mRNA) Normal Flow Ischemia/Reperfusion * 4 * Relative Fold Induction 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
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