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Nanoparticles for Drug Delivery
Nanoparticles for Drug Delivery
• 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
Methods of Drug 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)
~$200 Million in Ambisome sales in 2003.
Examples of Nanoparticles for Drug Delivery
Applications: Non-resistant cancers
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)
Examples of Nanoparticles for Drug Delivery Nanoparticles for Drug Delivery
Applications: Intracellular Infections
• Introduction – Systemic Drug Delivery
Macrophages infected with Salmonella incubated with ampicillin-loaded PACA nanospheres
Antibiotic-loaded nanospheres Resistance of many microorganisms to
antibiotics is often related to low uptake of antibiotics or reduced activity in acidic pH of lysosomes.
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.
Vauthier, et al. Adv. Drug Del Rev v55:519-548
(2003)
• Nanoparticles
• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery
Drug Carrier Systems
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
Nanoparticles for Drug Delivery
• Metal-based nanoparticles • Lipid-based nanoparticles • Polymer-based nanoparticles • Biological nanoparticles
Metal Nanoparticles for drug delivery Metal Nanoparticles
Generation
Size Definition Examples
First
> 1 µm
Able to release a drug at the target site but needing a particular type of administrat ion
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
Metal Nanoparticles Lipid-based nanoparticles for Drug Delivery
DOPE
DSPC
DOTAP
Note: direct tissue injection
Lipid-based nanoparticles for Drug Delivery
Deliverables:
-Small molecules (Amphotericin B, Daunorubicin, Dox orubicin – all approved and marketed drug formulations) Gilead, A lza
-Viruses and bacteria (as vaccines) – in developmen t
-Nucleic acids – in development
Lipid-based nanoparticles for Drug Delivery 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
Lipid-based nanoparticles for Drug Delivery
Drug properties and Liposome association
Hydrophilic Retained in aqueous interior Slowly rel eased over
**may be difficult to get several hours-several day s high loading
Hydrophobic Inserted into hydrophobic Excellent ret ention
interior of the liposome bilayer **can disrupt liposome at high concentrations
Intermediate Rapidly partition between Rapid releas e from liposomes
lipid bilayer and aqueous but pH manipulation or phase formation of molecular
complexes can result in good retention
Polymer-based nanoparticles for Drug Delivery • Poly(alkylcyanoacrylates) used extensively for tissue
adhesives for skin wounds and surgical glue (late 1960’s)
• Application as drug nanoparticulate carriers (1980s)
• For detailed review, see:
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Polymer-based nanoparticles for Drug Delivery Polymer-based nanoparticles for Drug Delivery
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Polymer-based nanoparticles for Drug Delivery 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)
Vauthier, et al. Adv. Drug Del Rev v55:519-548 (2003)
Biological nanoparticles --Viruses Biological nanoparticles --Viruses
Stratagene, Inc.
From an excellent review by Thomas et al. (2003) Nature v4:346
Biological nanoparticles --Viruses Nanoparticles for Drug Delivery
• 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 Br ownian motion
cause particle flocculation
2. Stabilization of colloids by electrostatic st abilization (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 potentia l:
Stability of Colloids in Physiological Environments
Aggregation of Colloids
A
-higher ionic strengths collapse this boundary layer
water 150 mM salt
M. Nikolaides
-at high salt concentration, no stable region -physiologic salt concentration ~150 nm
-- in intravenous delivery, this can be a major cau se of toxicity
Stability of Colloids in Physiological Environments
3. Stabilization of colloids by steric stabilizat ion: -- Add polymers to the surface of particles to prev ent the particles from
coming in close proximity to each other. At these distances, there is not enough attractive force for flocculation to occ ur.
--Note that this phenomena is solvent dependent (af fects the structure and interaction of the surface polymers)
Steric Stabilization
Steric Stabilization Dispersion Stabilization Choi, et al. J Disp Sci Tech (2003) v24:415
Steric Stabilization Steric Stabilization
Choi, et al. J Disp Sci Tech (2003) v24:415 Ogris et al. (1999) Gene Therapy 6:595 (1999).
Liposome Stability Liposome Stability Optimization
•Chemical stability
•Reducing oxidation – addition of antioxidants, stor age 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
• Nanoparticles
• Challenge 1: Stabilization • Challenge 2: Extended Circulation • Challenge 3: Targeting
• Examples :
Liposomes for chemotherapeutic delivery Cyclodextrin particles for gene delivery
Mechanisms of Removal from Circulation • 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
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
Kupffer Cells
http://education.vetmed.vt.edu/curriculum/VM8054/La bs/Lab20/Examples/exkupff.htm
Drug Carrier Systems: Influence of Physicochemical Properties Biodistribution of PEGylated Polymer-based Nanoparticles
1000 nm
6000 nm
500 nm
200 nm
10,000 nm
“+”-charged
“-”-charged
neutral
PEGylated
Non- PEGylated
Ogris et al. (1999) Gene Therapy 6:595 (1999).
Biodistribution of Lipid-based Nanoparticles PEGylation
Non-PEGylated liposomes
Why is there a dose-dependence?
PEGylated liposomes
Nanoparticles for Drug Delivery • 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
Allen, TM and Stuart, DD. “Liposome Pharmacokineti cs” In Liposomes Ed. Janoff, AS (2000)
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)
from Polymeric Biomaterials (2002), 2nd Ed., Edited by S. Dumitriu
Non-specific Targeting
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
Targeting Nanoparticles for Drug Delivery • 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
Examples of Nanoparticles for Drug Delivery
DOXIL Efficacy
Doxil (n = 118) Topotecan (n =
119) Time to progression
18.4 wks 18.3 wks
ORR 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 *Expressed as percentage.
**p = .01. TTP Plat Res = time-to-progression platinum resistant; TTP Plat Sens = time-to-progression platinum sensitive; OS = overall survival.
Examples of Nanoparticles for Drug Delivery Examples of Nanoparticles for Drug Delivery
DOXIL 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
DOXIL Pharmacokinetics
Rats Dogs
Gabizon, et al. Pharm Res v10:703 (1993)
Examples of Nanoparticles for Drug Delivery
DOXIL Toxicity
•Mild white blood cell depression •Skin toxicity (unique to Doxil) with full recovery. (Long distribution time?) •Cardiac toxicity – insignificant up to 1500 mg/m 2; 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-limiti ng toxicity
Examples of Nanoparticles for Drug Delivery
DOXIL Future improvements?
H
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ight
Uni
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Bac
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und
Po
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-Lys
ine
Su
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(Den
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4 P
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HO
HO
O
IDEAL SYSTEMIC DELIVERY VECTOR CYCLODEXTRIN POLYMER
• Non-toxic vehicle OH
O
OH O
O OH
HO O O OH
O
• Non-immunogenic O O
HO OH HO
OH O
OH OH
O
O HO
HO O
OH OH HO
HO O
OH
OH HO S
N O O
NH2
• Condensation of DNA OH HO
S O HO
O
(CH2)6 N
S NH2 2
OH HO O
OH HO
S + NH +
O HO OH HO
O O OH
O OH HO X O
HO
• Intracellular delivery • Targeting ligand
H2N O O
O OH
HO
• Stabilization of particles
• 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
(a)
1.00E+08
1.00E+07
BHK-21
CHO-K1
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
(b)
1.00E+00
200 nm
Gonzalez et al. Bioconjugate Chemistry (1999) v10:1 068-1074
TOXICITY COMPARISON TO BHK-21 CELLS INCLUSION COMPLEX FORMATION
Polymer PEI IC50=23µM
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
L
L
NEW METHOD OF SURFACE MODIFICATION TEM IMAGES OF PARTICLES
Stabilized Particle
L
L L
L L Pre-formed Particle
L L L
L L
L L L
A B A. Polyplexes in water B. PEGylated polyplexes
in water
C. Polyplexes in 50 mM
NaCl
D. PEGylated polyplexes
C D in 50 mM NaCl
Space bar is 100 nm
except in C where it
is 1000 nm
Targeted, Stabilized Particle Pun and Davis Bioconjugate Chemistry (2002 ) v13:6 30
Pun and Davis Bioconjugate Chemistry (2002 ) v13:6 30
Targeted Particles Biodistribution
Nucleic acid delivery Nanoparticle delivery
Pun, et al. Canc Biol Ther (in press) Pun, et al. Canc Biol Ther (in press)
Tumor uptake
Nucleic acid delivery Nanoparticle delivery
Pun, et al. Canc Biol Ther (in press)