Nano-Vehicular Targeted Drug Delivery

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Nano-Vehicular Targeted Drug Delivery

Nano-Vehicular Targeted Drug Delivery

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  • 1.
  • 2.
    • Drug Delivery
    In vast majority cases, drugs are delivered throughout the body via circulatory system
    Example : Intravascular injection or oral ingestion
  • 3.
    • Targeted Drug Delivery
    Method of delivering medication preferentially
    – To the right place in a body
    – At the right time, the right dose
    – For the right time period
    – Not to the other places where drug side-effects may arise
  • 4. Surgery
    • Surgical excision of cancer tissues
    • Accompany pain, risk of infection, poor wound healing
    Radiation
    • Method of completely killing cancer cells or shrinking tumors
    or relieving symptoms using high-energy radiation
    • Harm health tissues and damage nearby normal cells
    • Accompany pain, nausea, hair loss, damage to normal cells
    Chemotherapy
    • Chemical method of killing cells that divide rapidly (cancer cells)
    • Kills normal cells that divide rapidly under normal conditions
    • Accompany pain, nausea, memory loss, malnutrition
  • 5. Target to
    tumor cell
    Signaling
    Agent
    Targeting
    Agent
    Monitor
    targeting
    Drug
    Smuggle attached drug into tumor cell
    Released inside tumor cell
    Targeted Drug Delivery Systems
    • Nano-particle that carries attached drug to the site of action, with its path monitored by attached fluorescent detecting agent
    Tumor
    Cell
    Blood stream
    Technical Advantages
    • Higher efficacy resulting from selectively targeting
    and killing cancer cells
    • Reduced toxicity and lower side-effects
    • Potentially more cost-effective
    • Prospect for shorter treatments times
  • 6. Dendrimers : Ideal Building Block
    for Creating a Biologically Active Nano-material
    Dendrimers : Repeatedly Branched Molecules
    • Consist of a series of chemical shells built on a small core molecule
    • Each shell (generation) consists of monomer layers, made by repeating chemical-linking
    • Beyond G5 : Begin to become spherical and 3-D structure
  • 7. Surface Groups
    • Can be variously functionalized
    • Cationic / Anionic / Neutral
    • Targeting groups
    • Dyes & Biomarkers
    Similarity to Protein
    • Size / Weight
    • Very well-defined chemical structure
    • Ease of cellular uptake
  • 8. Targets : Folic Acid (FA)
    • FAR (high affinity receptor for FA) : over-expressed in several human cancers, even up to a 100-fold
    • Easily available and inexpensive / small molecular size
    Therapeutic Agent : Methotrexate (MTX)
    • Widely used chemotherapeutic drug for the treatment of a variety of malignancies
    • Inhibits cytosolic enzyme dihydrofolatereductase (DHFR)
    • Results in depletion of reduced FA required for nucleotide synthesis
    • Thus leading to the inhibition of DNA replicationand subsequent cell death
    Fluorescence Tag
    • Various chemicals : Fluorescein, AlexaFluor
    • Used for monitoring and tracking
  • 9. Fluorescent tagged nanodevice are taken up through
    the receptors then spread into cytosolic area
    To Target Specifically to Cancer Cells
    • Cells have on their surfaces receptors for specific molecules
    • Specific receptors for specific molecules are targeted
    • Specificity can be precisely controlled by targeting active receptor
    • Taken up into the cell through specific receptor on cancer cell
  • 10. Difficult to Assemble Multiple Functions onto One Dendrimer
    • Complex chemistry needed for self-assembly of core and shell dendrimers
    • No specificity between the coupling of dendrimers
    Self-Assembly using DNA
    • Each of two dendrimers carries single-stranded DNA with the same length
    • DNA strands are complementary
    • Self-assembly with forming double-stranded DNA
    • Barbell-shaped, two-dendrimer complexes
    • Fluorescence can be separated to the other
  • 11. Preparation of Each DNA-Dendrimer Conjugate
    1. Control surface charge density of amines
    - By substituting with acetyl groups to prevent infinite network formation due to electrostatic interaction
    - Acetylation limited to 90% of amines due to densely packed structure
    - G5 : 12 amine groups / G7 : 108 amine groups
    2. Prepare DNA strand
    - 16-32 nucleotides for spacer from dendrimer / 34 for complementary base pairing
    3. EDC/imidazole (0.1M) chemistry used : to activate DNAs for 10mins
    4. Slowly mix with LiCl (0.5M) used to weaken electrostatic interactions
    5. Allow to react overnight at RT
    6. Remove small molecules with membrane filter
    7. Purify non-conjugated DNA from using gel electrophoresis
    8. Extract purified DNA-dendrimer conjugate from gel
    9. Each dendrimer is functionalized : Target, drug and Fluorescence
  • 12. G5
    G5
    G7
    G7
    Annealing
    Self-assembly
    1. Mix two DNA-conjugated G7 and G5 dendrimers in equimolar ratio
    - to prevent crosslinking and formation of very large complexes
    2. Annealing : Heated at 90°C for 10 mins with hybridization buffer
    3. Cool at RT for 3 hours