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  1. 1. Drug and Gene Delivery Vehicles Drug and gene delivery vehicles are synthetic or biological biocompatible devices capable of carrying physiologically beneficial components in physiological environment. Features: 1. Improved bioavalability 2. Improved pharmacokinetics and pharmacodynamics 3. Site specificity 4. Decreased systemic toxicity 5. Designed to mimic natural carriers such as viruses and lipoproteins Focus: Synthetic Drug and Gene Delivery Vehicles
  2. 2. Block Copolymers Biocompatible heteropolymers comprising homopolymerc subunits that differ in hydrophobicity and functional groups. Major Characteristics: HLB (hydrophobic-lipophilic balance) CMC (critical micellar concentration) CMT (critical micellar temperature) HLB = 20 * Mh / M (Mh: weight of hydrophilic part, M: total weight) Example: Pluronic block copolymer JOURNAL OF CONTROLLED RELEASE Volume: 82 Issue: 2-3 Pages: 189-212
  3. 3. Mechanism of Drug Release from Micelles (A) disintegration of the micelles below CMC; (B) release of the drug as a result of partitioning. Relationship between the partitioning coefficients of pyrene and CMC in Pluronic systems
  4. 4. Preparation of micellar drug delivery vehicles Colloids and Surfaces B: Biointerfaces 16 (1999) 3–27
  5. 5. Direct dissolution Method: Block-copolymer is simply added to water or PBS. Co-dissolution Method: 1. Drug and block copolymer are first dissolved in a common organic solvent and then the phases are reversed by slow addition of the aqueous component. 2. The residual organic solvent is removed by dialysis or evaporation. Alternative: solvent from a common organic solution of the drug and block copolymer evaporated to give dry film. This film is re-dispersed in aqueous medium. Micellar drug formulation JOURNAL OF CONTROLLED RELEASE Volume: 82 Issue: 2-3 Pages: 189-212
  6. 6. Temperature-Sensitive Polymers
  7. 7. pH-Sensitive Polymers as Drug Carriers Diblock copolymers that formed two types of micelles in aqueous solution depending on pH. These resulting states were described as ‘schizophrenic’ since by changing external pH, temperature or ionic strength the more hydrophilic block could be transformed to a hydrophobic state in order to form the core of a micelle. The use of poly(4-vinylbenzoic acid) (pKa 5 7.1) as one block and poly(2-N- (morpholino)ethylmethacrylate) (pKa of the conjugate acid 5 4.9) ensured that precipitation did not occur during pH variation across the isoelectric point. Chem. Soc. Rev., 2005, 34, 276–285
  8. 8. Architectures of responsive hydrogels
  9. 9. Responsive DNA Carrier Poly(N-isopropylacrylamide) protects DNA from degradation by endonuclease at 37 o C but not at 27 o C
  10. 10. Responsive Hydrogel System for Insulin Release Hydrogel was prepared of an insulin-containing reservoir within a poly(methacrylic acid graft-poly[ethylene glycol]) (P(MAA-g-EG)) copolymer in which glucose oxidase was immobilised. Ingress of glucose through the polymer layer to the entrapped glucose oxidase resulted in a pH drop as glucose was oxidised to gluconic acid, and the released protons caused the pendent PMAA chains of the hydrogel to contract, thus opening the gates to allow insulin transport. Chem. Soc. Rev., 2005, 34, 276–285
  11. 11. Biopolymers and artificial polypeptides Progress in Polymer Science Volume 29, Issue 12, December 2004, Pages 1173-1222 Tri-block copolymer consists of 230 amino acids, 84 of which make up the helix repeat and 90 of which make up the alanyl glycine rich repeat.
  12. 12. Liposomes Targeted Drug and Gene Delivery Natural phospholipids, pegylated phospholipids, synthetic block copolymers Preparation: Dialysis, High pressure extrusion, sonication. Control over the size lamellarity and contents
  13. 13. “Evolution” of Liposomes A. Early traditional phospholipids ‘plain’ liposomes with water soluble drug. B. Antibody-targeted immunoliposomes. C. Long-circulating liposome grafted with a protective polymer. D. Long-circulating immunoliposomes. E. New-generation liposomes. Nature Rev. Drug Discov. 2005, 4, 145-160
  14. 14. Chemistry of ligand attachments Nature Rev. Drug Discov. 2005, 4, 145-160
  15. 15. Interactions of Liposomes with Cells Nature Rev. Drug Discov. 2005, 4, 145-160
  16. 16. Fusogenic and stimuli-sensitive liposomes Nature Rev. Drug Discov. 2005, 4, 145-160
  17. 17. Immunoliposomes in action Nature Rev. Drug Discov. 2005, 4, 145-160
  18. 18. Liposomes in Diagnostic Imaging Nature Rev. Drug Discov. 2005, 4, 145-160
  19. 19. Liposomes for Gene Delivery • Cationic Lipids are Required to bind DNA into Lipoplex Often, lipids are designed to decay in the endosomes, allowing DNA escape Stable lipids like DOTAP do not provide efficient cell transfection J. Med. Chem., 50, 4269-4278, 2007
  20. 20. pH-dependent release of contents
  21. 21. Gene Delivery by Cationic Lipoplexes in vivo Barriers for intravenous gene delivery. DNA-containing NP are injected intravenously into human body. Serum proteins may bind to the particles, crosslink them and increase the particle size. This can result in rapid particle elimination (Insert 1). The Kupffer cells may take up particles and decrease their access to the hepatocytes (Insert 2). Circulating NP may extravasate in tumor tissue through the leaky tumor vessels (Insert 3). Particles then need to pass through the crowded extracellular matrix to contact the cell surface (Insert 4). When the particles are internalized into cells, DNA must escape from the endosome and find its way into the nucleus (Insert 5). Pharmaceutical Research, 2007, 24, 438-449
  22. 22. Optimizing conditions: Pharmaceutical Research, 2007, 24, 438-449
  23. 23. Modifying Lipolexes: • 1. Cationic headgroups tethered to the lipid via disulfide linker • 2. Lipoplexes formulated under optimized conditions • 3. External cationic headgroups exchanged for anionic headgroups • 4. cell-binding TAT peptide, sequence GRKKRRQRRRGYG, attached Outcome: Up to 80-fold increase in the transfection efficiency MOLECULAR THERAPY, 2005, 409-417 N S S N + CH2Cl2 MeOH H H H S SN H N SH Cl H H H N S S MeI H H H N S S I H H H HS NaOH Scheme 6 Reducible cationic cholesterol 16 17 18 42.5% 95%
  24. 24. Manipulation with cationic lipids using transmembrane diffusion potential O O N H O NH O O O O N H O O NH ON H O O O O O N H O O O Valinomycin K+ -selective Carrier O O NHBu O O NHBu O O NHBu Triphenoxyacetamide Cl- -selective transporter J. Am. Chem. Soc. 125, 2003, 2840.
  25. 25. Towards Improved Non-Viral Gene Therapy K+ SO4 2- K+ - - - - - - - - - + ++ + + + + + Li+ Cl- Cl- Li+ Chem. Commun. 2007, 383. ∆ Ψ flip valinomycin K+ SO4 2- K+ - - - - - - - - - + ++ + + + + + Li+ Li+ Cl- Cl- triphenoxyacetamide Application of valinomycin and triphenoxyacetamide to K2SO4 liposomes suspended in LiCl resulted in a rapid formation of stable negative potential (~ -360 mV)
  26. 26. How to test lipid flip Lipid flipping in cells is due to flippases (part of the apoptotic cycle) Standard assay: fluorescence of NBD-PE is quenched with 60 mM Na2S2O4 Invasive quencher, no real-time monitoring of the flippase activity Non-invasive methodology is required Bioconjugate Chem. 18, 2007, 1507-1515
  27. 27. Test of Flip (Quenching Experiments) 0.00E+00 2.00E+06 4.00E+06 6.00E+06 8.00E+06 1.00E+07 1.20E+07 0 500 1000 1500 2000 2500 Time Fluorescence Blank 5 min 2 hrs 12 hrs Na2S2O4 Triton Chem. Commun. 2007, 383.
  28. 28. Improved Stability of Cationic Liposomes in the Presence of Human Serum Albumin DLS Study of Interaction with HSA 0 1000 2000 3000 0.00 0.10 0.20 0.30 0.40 Time (hours) Diameter(nm) 15 mol% Ethyl PC/EYPC in LiCl buffer without ionophores 15 mol% Ethyl PC/EYPC in LiCl buffer with ionophores 100% EYPC Control Chem. Commun. 2007, 383.
  29. 29. Dendrimers Dendrimers are a class of well-defined nanostructured macromolecules that possess narrow mass or size polydispersity and tree-like architecture distinguished by exponential numbers of discrete dendritic branches radiating out from a common core. Synthesis: 1. Divergent 1979 2. Convergent 1989 3. Self-Assembly 1996 4. “Lego” Chemistry 2003 5. “Click” chemistry 2004 Drug Discovery Today, Volume 15, Issues 5-6, March 2010, Pages 171-185
  30. 30. Nomenclature of Dendrimers Polypropylene imine dendrimer (G5) Chem. Soc. Rev., 2004, 33, 43-63
  31. 31. Synthesis Divergent: Convergent: Chem. Soc. Rev., 2004, 33, 43-63
  32. 32. Self Assembly J. Am. Chem. Soc., 2002, 124 (46), pp 13757–13769
  33. 33. Click Chemistry Current Topics in Medicinal Chemistry, 2008, 8, 1294-1309
  34. 34. Lego, or Orthogonal Coupling Current Topics in Medicinal Chemistry, 2008, 8, 1294-1309 The orthogonal coupling strategy is based on the use of two monomers of the type AB2 and CD2, for example, which are designed such that the focal functionalities A and C of each monomer react, respectively, only with the branching points D and B.
  35. 35. Dendrimers as drug delivery vehicles Drug Discovery Today, Volume 15, Issues 5-6, March 2010, Pages 171-185
  36. 36. Drug Delivery by Dendrimers Schematic representation of the G = 4; PAMAM (polyamidoamine) dendrimer linked to N-acetylcysteine by disulfide bonds. The dendrimer delivers the drug intracellularly by the cleavage of the disulfide linkage owing to the thiol exchange redox reactions initiated by the intracellular glutathione. The dendrimer carrier is then excreted by the cell.
  37. 37. Anticancer: Cisplatin: 3-15-fold drop in toxicity. Bioavailability increased in vitro and in vivo. 5-fluorouracil: Improved PK and PD. Bioavailability increased in vitro and in vivo. Doxorubicin: Bioavailability increased, anticancer activity decreased. DNA carrier Antibody Carrier Drug Carrier (ibuprofen has shown 40-fold increase in bioavailability) MRI contrast agents carrier Examples Current Topics in Medicinal Chemistry, 2008, 8, 1294-1309
  38. 38. MRI Contrast agent carrier: Drug Discovery Today, Volume 15, Issues 5-6, March 2010, Pages 171-185
  39. 39. Magnetic Nanoparticles (Iron Oxide) Suitable for: 1. Magnetic resonance imaging contrast enhancement 2. Tissue repair 3. Detoxification of biological fluids 4. Drug Delivery 5. Cell Separation For these applications, the particles must have combined properties of high magnetic saturation, biocompatibility and interactive functions at the surface. The surfaces of these particles could be modified through the creation of layer of organic polymer or inorganic metallic (e.g. gold) or oxide surfaces (e.g. silica or alumina), suitable for further functionalization by the attachment of various bioactive molecules. Biomater. 2005, 26, 3995-4021
  40. 40. Preparation of Nanoparticles Chemistry AOT: Dioctyl sodium sulfosuccinate Micellar Reactors
  41. 41. Surface modification of for biomedical applications and their effect on stability and magnetization Large magnetic nanoparticles form aggregates due to hydrophobic nature and magnetization. Smaller and more uniform nanoparticles can be prepared inside the aqueous droplets of reverse micelles. Biomater. 2005, 26, 3995-4021
  42. 42. Typical coatings for magnetic nanoparticles Polyethylene glycol (PEG) Improves biocompatibility, circulation time and internalization Dextran Enhances circulation time, stabilizes the colloidal solution Fatty acids Colloidal stability, terminal functional carboxyl groups Polyacrylic acid Helps in bioadhesion Polypeptides Good for cell biology, e.g. targeting to cell Chitosan Suitable for gene delivery Polyisopropylacryl-amide Termosensitive Drug Delivery Biomater. 2005, 26, 3995-4021
  43. 43. Modification of surface with Ligands Transferrin targets tranferrin receptors in cancer cells Nerve growth factor promotes nerve growth Ceruloplasmin copper carrier, antioxidant Tat-peptide enhances intracellular delivery RGD peptide Increases cell spreading and differentiation Folic acid Preferentially target cancer cells lactoferrin Binds to fibroblasts Biomater. 2005, 26, 3995-4021
  44. 44. Applications of magnetoparticles in detail: Tissue repair Tissue repair using iron oxide nanoparticles is accomplished either through welding, or through soldering. Temperatures greater than 50 °C are known to induce tissue union. Particles are heated either by absorption of light or by the magnetic field. Core-shell particles covered with gold are useful due to strong absorption of light. Biomater. 2005, 26, 3995-4021
  45. 45. Applications of magnetoparticles in detail: Drug Delivery Particles of the size between 10-100 nm are used. For improved circulation, coated with PEG. PEG enhances uptake of particles by tumor cells. Can also serve for targeting erythrocites by magnetic field. There is indication that magnetic nanoparticles can cross BBB. Liposomes with magnetic particles have been prepared. These liposomes can be controlled by magnetic field.
  46. 46. Applications of magnetoparticles in detail: Hyperthermia An old method of treatment of malignant tumors Magnetic induction hyperthermia, means the exposition of cancer tissues to an alternating magnetic field. Magnetic field is not absorbed by the living tissues and can be applied to deep region in the living body. When magnetic particles are subjected to a variable magnetic field, some heat is generated due to magnetic hysteresis loss. The amount of heat generated depends on the nature of magnetic material and of magnetic field parameters. Very small amounts of magnetic fine particles in the order of tenth of milligram may easily be used to raise the temperature of biological tissue locally up to cell necrosis. Differential endocytosis of modified aminosilan magnetite nanoparticles into primary glioblastoma cells, but not in normal glial cells in vitro, has been reported. Heat-induced therapeutic gene expression. Biomater. 2005, 26, 3995-4021
  47. 47. Applications of magnetoparticles in detail: Magnetofection Magnetofection (MF) is a method in which magnetic nanoparticles associated with vector DNA are transfected into cells by the influence of an external magnetic field. Cationic coating of nanoparticles is required. Due to inherent permeability of nanoparticles to the cells, up-to 360-fold increase in transfection has been reported. Conclusions: Magnetic nanoparticles can be prepared with high degree of control over the size and surface coating. They can be targeted by receptors on the surface or by external magnetic field. Magnetic particles can assist both drug and gene delivery. They provide independent therapeutic path through hyperthermia
  48. 48. Summary Four different types of synthetic drug delivery vehicles have been discussed: Micellar and hydrogel systems comprising block copolymers Liposomes Dendrimers Magnetic nanoparticles Methods of preparation were outlined, and biopharmaceutical applications of these vehicles have been discussed.