Presentation

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. 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 * M
h
/ M (M
h
: weight of hydrophilic part, M: total weight)
Example: Pluronic block copolymer
JOURNAL OF CONTROLLED RELEASE Volume: 82 Issue: 2-3 Pages: 189-212

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. Preparation of micellar drug delivery vehicles
Colloids and Surfaces B: Biointerfaces 16 (1999) 3–27

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. Temperature-Sensitive Polymers

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. Architectures of responsive hydrogels

9. Responsive DNA Carrier
Poly(N-isopropylacrylamide) protects DNA from degradation by
endonuclease at 37
o
C but not at 27
o
C

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. 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. 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. “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

15. Chemistry of ligand attachments
Nature Rev. Drug Discov. 2005, 4, 145-160

16. Interactions of Liposomes with Cells
Nature Rev. Drug Discov. 2005, 4, 145-160

17. Fusogenic and stimuli-sensitive liposomes
Nature Rev. Drug Discov. 2005, 4, 145-160

18. Immunoliposomes in action
Nature Rev. Drug Discov. 2005, 4, 145-160

19. Liposomes in Diagnostic Imaging
Nature Rev. Drug Discov. 2005, 4, 145-160

20. 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

21. pH-dependent release of contents

22. 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

23. Optimizing conditions:
Pharmaceutical Research, 2007, 24, 438-449

24. 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
SN
+
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%

25. 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
OO
Valinomycin
K
+
-selective Carrier
O
O NHBu
O
O NHBu
O
O NHBu
Triphenoxyacetamide
Cl
-
-selective transporter
J. Am. Chem. Soc. 125, 2003, 2840.

26. Towards Improved Non-Viral Gene Therapy
K
+
SO4
2-
K
+
-
-
--
-
-
-
-
-
+
++
+
+
+
+
+Li
+
Cl
-
Cl
-
Li
+
Chem. Commun. 2007, 383.
D
Yflip
valinomycin
K
+
SO
4
2-
K
+
-
-
--
-
-
-
-
-
+
++
+
+
+
+
+
Li
+
Li
+
Cl
-
Cl
- triphenoxyacetamide
Application of valinomycin and triphenoxyacetamide to K
2
SO
4
liposomes suspended in
LiCl resulted in a rapid formation of stable negative potential (~ -360 mV)

27. 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 Na
2
S
2
O
4
Invasive quencher, no real-time monitoring of the flippase activity
Non-invasive methodology is required
Bioconjugate Chem. 18, 2007, 1507-1515

28. 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 1000150020002500
Time
F
l
u
o
r
e
s
c
e
n
c
e
Blank
5 min
2 hrs
12 hrs
Na
2
S
2
O
4
Triton
Chem. Commun. 2007, 383.

29. 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)
D
i
a
m
e
t
e
r

(
n
m
)
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.

30. 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

31. Nomenclature of Dendrimers
Polypropylene imine dendrimer (G5)
Chem. Soc. Rev., 2004, 33, 43-63

32. Synthesis
Divergent:
Convergent:
Chem. Soc. Rev., 2004, 33, 43-63

33. Self Assembly
J. Am. Chem. Soc., 2002, 124 (46), pp 13757–13769

34. Click Chemistry
Current Topics in Medicinal Chemistry, 2008, 8, 1294-1309

35. 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 AB
2
and CD
2
, 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.

36. Dendrimers as drug delivery vehicles
Drug Discovery Today, Volume 15, Issues 5-6, March 2010, Pages 171-185

37. 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.

38. 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

39. MRI Contrast agent carrier:
Drug Discovery Today, Volume 15, Issues 5-6, March 2010, Pages 171-185

40. 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

41. Preparation of Nanoparticles
Chemistry
AOT: Dioctyl sodium sulfosuccinate
Micellar Reactors

42. 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

43. 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

44. 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

45. 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

46. 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.

47. 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

48. 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

49. 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.