Nanoparticles for Drug Delivery
Nanoparticles for Drug Delivery
• Introduction – Systemic Drug Delivery
• Nanoparticles
Suzie Pun
Dept of Bioengineering, University of Washington
Frontiers in Nanotechnology
May 18, 2004
Methods of Drug Delivery
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Advantages of Nanoparticles for Drug Delivery
• Oral Delivery
• Inhalation
• Transdermal
• Implantation
• Injection
University of Illinois, Urbana-Champagne
Examples of Nanoparticles for Drug Delivery
Liposomal Amphoterin, sold by Gilead (Ambisome) and Enzon (Abelcet)
Examples of Nanoparticles for Drug Delivery
Applications: Non-resistant cancers
~$200 Million in Ambisome sales in 2003.
Gilead Sciences
•
Amphotericin treats fungal and parasite infection
•
Most commonly used in patients with depressed white blood cell count
(cancer and chemotherapy patients, HIV-infected patients, elderly patients).
•
Liposomal formulation is preferred because of decreased side effects and
prolonged drug exposure (due to slow release)
•In hepatic metastases model, the reduction in number of metastases was
greater with Dox-loaded nanospheres than free dox. (Why hepatic model?)
•No special affinity for tumor tissue detected. Most nanospheres located within
Kupffer cells. See proposed mechanism of action
•Note that this approach reduces side effects and toxicity! Small molecules are
distributed throughout the body.
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
1
Examples of Nanoparticles for Drug Delivery
Applications: Intracellular Infections
Nanoparticles for Drug Delivery
• Introduction – Systemic Drug Delivery
Antibiotic-loaded nanospheres
• Nanoparticles
Resistance of many microorganisms to
antibiotics is often related to low uptake of
antibiotics or reduced activity in acidic pH of
lysosomes.
Macrophages infected with
Salmonella incubated with
ampicillin-loaded PACA
nanospheres
1. Ampicillin-loaded nanospheres for Listeria
treatment. Dramatic improvement over free
drug; bacterial counts in liver reduced at
least 20-fold.
2. Ampicillin-loaded nanospheres for
Salmonella treatment. Drug alone – required
32 mg per mouse; with nanoparticle, only 0.8
mg ensured survival.
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Drug Carrier Systems
Nanoparticles for Drug Delivery
• Metal-based nanoparticles
Generation
Size
Definition
Examples
First
> 1 µm
Able to release a drug at the target site but
needing a particular type of administration
Microspheres and microcapsules for
chemoembolization
Second
< 1 µm
Carriers that can be given by a general
route able to transport a drug to the target
site
Liposomes, nanoparticles, polymer drug
carriers
Third
< 1 µm
Carriers able to recognize a specific target
Monoclonal antibodies; second-generation
carriers with targeted antibodies or other
ligands
• Lipid-based nanoparticles
• Polymer-based nanoparticles
• Biological nanoparticles
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
Metal Nanoparticles for drug delivery
Metal Nanoparticles
2
Lipid-based nanoparticles for Drug Delivery
Metal Nanoparticles
DOPE
DSPC
DOTAP
Note: direct tissue injection
Lipid-based nanoparticles for Drug Delivery
Lipid-based nanoparticles for Drug Delivery
Deliverables:
-Small molecules (Amphotericin B, Daunorubicin, Doxorubicin – all
approved and marketed drug formulations) Gilead, Alza
-Viruses and bacteria (as vaccines) – in development
-Nucleic acids – in development
Many formulation methods -1. Mixing lipids together in
organic solution.
2. Remove solvent by
evaporation
3. Hydration with aqueous
solvent containing drug to
form multilamellar vesicles
4. Sonication or extrusion are
common methods to reduce
the size of the liposomes
Avanti Polar Lipids
Polymer-based nanoparticles for Drug Delivery
Lipid-based nanoparticles for Drug Delivery
Drug properties and Liposome association
Hydrophilic
Hydrophobic
Intermediate
Retained in aqueous interior
**may be difficult to get
high loading
Slowly released over
several hours-several days
•
Poly(alkylcyanoacrylates) used extensively for tissue
adhesives for skin wounds and surgical glue (late 1960’s)
Inserted into hydrophobic
interior of the liposome bilayer
**can disrupt liposome at
high concentrations
Excellent retention
•
Application as drug nanoparticulate carriers (1980s)
•
For detailed review, see:
Rapidly partition between
lipid bilayer and aqueous
phase
Rapid release from liposomes
but pH manipulation or
formation of molecular
complexes can result in
good retention
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
3
Polymer-based nanoparticles for Drug Delivery
Polymer-based nanoparticles for Drug Delivery
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Polymer-based nanoparticles for Drug Delivery
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Polymer-based nanoparticles for Drug Delivery
DEGRADATION OF NANOPARTICLES
Hydrolysis of ester bond; degradation products
(alkylalcohol and poly(cyanoacrylic acid) are
eliminated by kidney filtration.
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Biological nanoparticles --Viruses
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Biological nanoparticles --Viruses
Scientific American
Stratagene, Inc.
From an excellent review by Thomas et al. (2003) Nature
v4:346
4
Nanoparticles for Drug Delivery
Biological nanoparticles --Viruses
• Introduction – Systemic Drug Delivery
• Nanoparticles
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Thomas et al. (2003) Nature v4:346
Stability of Colloids in Physiological Environments
1. Attractive van der Waals forces and random Brownian motion cause
particle flocculation
Stability of Colloids in Physiological Environments
Aggregation of Colloids
2. Stabilization of colloids by electrostatic stabilization (DLVO theory):
-- Charged particles have a counter-ion layer. The charged surface and
counter-ion layer is called the “electrostatic double layer”. For
homogenous colloid suspensions, this electrostatic double layer
acts as a repulsive force between particles.
--The sum of the van der Waals force and the double layer repulsion
force gives the DVLO interaction potential:
A
C
-higher ionic strengths collapse
this boundary layer
water
150 mM salt
-at high salt concentration, no
stable region
-- in intravenous delivery, this can be a major cause of toxicity
-physiologic salt concentration
~150 nm
M. Nikolaides
Stability of Colloids in Physiological Environments
Steric Stabilization
3. Stabilization of colloids by steric stabilization:
-- Add polymers to the surface of particles to prevent the particles from
coming in close proximity to each other. At these distances, there is
not enough attractive force for flocculation to occur.
--Note that this phenomena is solvent dependent (affects the structure
and interaction of the surface polymers)
Steric Stabilization
Dispersion Stabilization
Choi, et al. J Disp Sci Tech (2003) v24:415
5
Steric Stabilization
Steric Stabilization
Ogris et al. (1999) Gene Therapy 6:595 (1999).
Choi, et al. J Disp Sci Tech (2003) v24:415
Liposome Stability
Liposome Stability Optimization
•Chemical stability
•Reducing oxidation – addition of antioxidants, storage at low
temperatures and pH 6.5
•Removal of water – spray drying or lyophilization (but both have
to occur under controlled and optimized conditions)
•Physical stability
•Electrostatic stabilization
•Steric stabilization
•Cream or hydrogel incorporation
Nanoparticles for Drug Delivery
• Introduction – Systemic Drug Delivery
Mechanisms of Removal from Circulation
•
• Nanoparticles
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Fast removal from circulation
-binding to cells, membranes, or plasma proteins
-uptake by phagocytes (macrophages)
-trapping in capillary bed (lungs)
•
Renal clearance
-size restriction for kidney glomerulus is ~30-35 kDa for polymers
(~20-30 nm)
•
Extravasation
-depends on the permeability of blood vessels
-capillaries are thought to be more permissive to extravasation
-note: for cancer applications this works to our advantage: EPR!
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
6
Drug Carrier Systems: Influence of Physicochemical Properties
•
Molecular weight
-macromolecules smaller than renal threshold are rapidly eliminated
-for larger, non-degradable molecules, excretion is useful to decrease
toxicity.
•
Charge
-positively-charged macromolecules will interact with cells and
membranes (remember the negatively charged proteoglycans?)
-negatively charged macromolecules are picked up by macrophages
such as Kupffer cells, that contain polyanion scavenger receptors on
their surface.
•
PEGylation
-may increase circulation by reducing non-specific protein binding.
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
Drug Carrier Systems: Influence of Physicochemical Properties
1000 nm
6000 nm
500 nm
Drug Carrier Systems: Influence of Physicochemical Properties
Kupffer Cells
http://education.vetmed.vt.edu/curriculum/VM8054/Labs/Lab20/Examples/exkupff.htm
Biodistribution of PEGylated Polymer-based Nanoparticles
“+”-charged
PEGylated
“-”-charged
neutral
200 nm
10,000 nm
NonPEGylated
Ogris et al. (1999) Gene Therapy 6:595 (1999).
Biodistribution of Lipid-based Nanoparticles
PEGylation
Non-PEGylated
liposomes
Nanoparticles for Drug Delivery
• Introduction – Systemic Drug Delivery
• Nanoparticles
Why is there a dose-dependence?
PEGylated
liposomes
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Allen, TM and Stuart, DD. “Liposome Pharmacokinetics”
In Liposomes Ed. Janoff, AS (2000)
7
Non-specific targeting: EPR Effect
•
Tumors generally can’t grow beyond 2 mm in size without becoming
angiogenic (attracting new capillaries) because difficulty in obtaining
oxygen and nutrients.
•
Tumors produce angiogenic factors to form new capillary structures.
•
Tumors also need to recruit macromolecules from the blood stream to
form a new extracellular matrix.
•
Permeability-enhancing factors such as VEGF (vascular endothelial
growth factor) are secreted to increase the permeability of the tumor
blood vessels.
•
This effect is called the “enhanced permeability and retention effect”
(EPR)
Non-specific Targeting
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
Targeting Ligands
Targeting
• Small Molecules
– Galactose/Glucose/Mannose
– Folate
• Peptides
– RGD
• Proteins
– Transferrin
– Antibodies
– LDLs
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endocytosis.html
Choi, et al. J Disp Sci Tech (2003) v24:415
Nanoparticles for Drug Delivery
Targeting
• Introduction – Systemic Drug Delivery
• Nanoparticles
• Challenge 1: Stabilization
• Challenge 2: Extended Circulation
• Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery
Cyclodextrin particles for gene delivery
Choi, et al. J Disp Sci Tech (2003) v24:415
8
Examples of Nanoparticles for Drug Delivery
DOXIL
Examples of Nanoparticles for Drug Delivery
DOXIL Proposed Mechanism of Action
Formulation
1. Doxorubicin-containing
core
2. Lipid Bilayer membrane
3. PEG-coated surface
4. <100 nm
Examples of Nanoparticles for Drug Delivery
Examples of Nanoparticles for Drug Delivery
DOXIL Toxicity
DOXIL Pharmacokinetics
Dogs
Rats
•Mild white blood cell depression
•Skin toxicity (unique to Doxil) with full recovery. (Long distribution
time?)
•Cardiac toxicity – insignificant up to 1500 mg/m2; Much lower toxicity
than doxorubicin (lower peak plasma level; decrease availability to
cardiac muscle)
•Hair loss – rare; only seen in ~6% of patients
•Mucositis – ulceration of oral mucosa. Dose-limiting toxicity
Gabizon, et al. Pharm Res v10:703 (1993)
Examples of Nanoparticles for Drug Delivery
DOXIL Efficacy
Doxil (n =
118)
Topotecan (n =
119)
18.4 wks
18.3 wks
CR
4*
5*
PR
16*
12*
TTP Plat Res
12.3 wks
6.5 wks
TTP Plat Sens
28.4 wks
28.8 wks
OSPlat Res
33.4 wks
37.3 wks
OSPlat Sens
86.1 wks**
63.3 wks
Time to
progression
Examples of Nanoparticles for Drug Delivery
DOXIL Future improvements?
ORR
*Expressed as percentage.
**p = .01.
TTP Plat Res = time-to-progression platinum resistant;
TTP Plat Sens = time-to-progression platinum sensitive;
OS = overall survival.
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IDEAL SYSTEMIC DELIVERY VECTOR
CYCLODEXTRIN POLYMER
OH
•
•
•
•
•
•
OH
Non-toxic vehicle
Non-immunogenic
Condensation of DNA
Intracellular delivery
Targeting ligand
Stabilization of
particles
HO
O
O
OH
O
OH HO
HO
O
O
HO
OH
O
OH
O
HO
HO
H
N
S
HO
O
S
NH2+
O
HO
O
(CH2)6
H
N
O
OH
O
OH
HO
O
HO
NH2
S
HO
OH
O
X
O
OH
O
OH
O
O
O
OH
OH HO
O
OH
S
NH2+
OH HO
O
O
HO
O
OH
H2N
HO
OH
O
HO
O
O
OH HO
O
O
OH
HO
HO
• Ave. MW 5.8 kDa, polydispersity index Mw/Mn = 1.12
• Degree of polymerization ~ 5 (10 charges/chain)
verified by end group analysis and light scattering
Gonzalez et al. Bioconjugate Chemistry (1999) v10:1068-1074
PARTICLE ASSEMBLY
TRANSFECTION COMPARISON
DNA
1.00E+08
(a)
BHK-21
1.00E+07
CHO-K1
Relative Light Units
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
HO
HO OH
HO
O
S
O
HO
O
O
H
N
(CH2)6
NH2+
H
N
OH
OH
OH
O
O
HO
HO OH
HO
O
O
O
1.00E+01
NH2
S
HO
OH
S
NH2+
X
O
O
O
HO
O
O
1.00E+00
OH
HO
HO
(b)
PEI
O
O
HO
OH
O
OH
CD-polymer 4
OH
O
O
O
OH
OH OH HO
Lipofectamine
HO
OH
O
HO
O
O
S
H2N
HO
OH
Superfect
(Dendrimer)
O
O
O
OH
O
OH HO
HO
Poly-L-Lysine
O
Background
OH
OH
HO
200 nm
Gonzalez et al. Bioconjugate Chemistry (1999) v10:1068-1074
TOXICITY COMPARISON TO BHK-21 CELLS
Polymer PEI
IC50=23µM
INCLUSION COMPLEX FORMATION
Lipid,
Lipofectamine
IC50=6.4 µM
+
Polycation
Superfect
IC50=11 µM
Cyclodextrin
polymer
IC50=320 µM
IC50 values reported as charge concentrations in the presence of
DNA
Association Constant is 104–105
10
TEM IMAGES OF PARTICLES
NEW METHOD OF SURFACE MODIFICATION
-Ad
P EG
A
B
C
D
A. Polyplexes in water
B. PEGylated polyplexes
in water
C. Polyplexes in 50 mM
NaCl
D. PEGylated polyplexes
in 50 mM NaCl
Stabilized Particle
L
Lig
Pre-formed Particle
L
an
d
- PE
L
GAd
L
L
L
L
L
L
Space bar is 100 nm
except in C where it
is 1000 nm
L
L
L
L
L
L
Targeted, Stabilized Particle
Pun and Davis Bioconjugate Chemistry (2002 ) v13:630
Targeted Particles
Pun and Davis Bioconjugate Chemistry (2002 ) v13:630
Biodistribution
Nucleic acid delivery
Pun, et al. Canc Biol Ther (in press)
Nanoparticle delivery
Pun, et al. Canc Biol Ther (in press)
Tumor uptake
Nucleic acid delivery
Nanoparticle delivery
Pun, et al. Canc Biol Ther (in press)
11